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  1. Last 7 days
    1. The activities ofβ-xylosidase, xylan acetylesterase and arbinofuranosidase were measured using 1 mM p-nitrophenylxylopyranoside, p-nitrophenylacetate and p-nitrophenylarabinofuranoside, respectively prepared in sodium citrate buffer (0.1 M, pH 7.0). One mL of reaction mixture containing 0.2 mL of crude enzyme solution, 0.3 mL of sodium citrate buffer (0.1 M, pH 7.0) and 0.5 mL of substrate was incubated at 80 °C for 30 min. The reaction was terminated by adding 2 mL sodium carbonate-bicarbonate buffer (1.0 M, pH 10.0). The activities were determined using p-nitrophenol standard curve (1-10 μg mL-1) drawn using absorbance values measured in spectrophotometer at 400 nm. One unit of the enzyme is defined as the amount of enzyme that liberates 1μmole of p-nitrophenol mL-1min-1 under assay conditions.
    2. Assays for β-Xylosidase, acetylesterase and arbinofuranosidase
    3. Metagenomic library obtained from various extracted DNA was screened by replica plating method on 0.3 % w/v RBB xylan containing LB-amp plates. The cells were allowed to grow for overnight at 37 °C and thereafter incubated at 4 °C till the appearance of zone of hydrolysis. A total of 36,400 clones from various environmental samples were screened.
    4. SCREENING OF THE TRANSFORMANTS FOR XYLANASE ACTIVITY
    5. PurifiedDNA fragments of size 2-8 kb were ligated to the treated vector using a 1:3::vector :insert ratio in a volume of 10 μL. The total amount of DNA was about 0.5 μg. Vector and insert DNA was heated to 45 °C for 10 min and the immediately chilled on ice for 5 min prior to addition of ligase and buffer. T4 DNA ligase (NEB, England) was added to a final concentration of 0.125 UμL-1 and reactions were incubated at 16 °C for overnight in a ligation chamber. Reaction mixture incubated under same condition without addition of the enzyme was used as control. A ligation reaction was also set up under condition with linear plasmid DNA containing the
    1. 5 III of the ligation mix was added to competent cells and mixed gently and the mix was kept on ice for 30 min before giving a heat shock at 42°C for 1 min. The· mixture was incubated on ice for 2 min and 900 III of LB broth was added to each tube. The cells were recovered by centrifugation at 250 rpm at 37°C for 1 h and were plated on LB agar plates containing the appropriate antibiotic(s) and incubated overnight at 37°C
    2. Transformation in E. coli
    3. . Jalciparum cultures were maintained as described previously (Trager and Jensen, 1976). Briefly, P. Jalciparum strain 3D7 was cultured at 37°C in RPMI " 1640 medium (list I) in 0+ RBCs supplemented with 10% AB+ human serum or : 0.5% Albumax II (complete medium). All media were preheated to 37°C and care was taken to minimize the handling time outside the 37°C incubator. Cultures were gassed with 5% CO2, 3% O2, and 92% N2 for 20 seconds and maintained at 37°C.
    4. Maintenance of P.falciparum cultures
    1. Trypan blue is a diazo vital stain which selectively colours the dead cells blue that can be visualized under light microscope. Equal volumes of cell suspension and -0.4% trypan blue dye were mixed and incubated at room temperature for 5 min. 10 J!L of stained cells were loaded on to a hemocytometer and a count of the number of viable and dead cells were made. This procedure was carried out routinely to ensure that cell viability is >95% before plating cells for experiments
    2. Assay for cell viability by Trypan blue dye exclusion method
    1. Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods (A. J. McCoy, 2007). The novel algorithms in Phaser are based on maximum likelihood probability theory and multivariate statistics rather than the traditional least-squares and Patterson methods. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise. One of the design concepts of Phaser was that it be capable of a high degree of automation. Phaser has novel maximum likelihood phasing algorithms for the rotation functions and translation functions in MR, but also implements other non-likelihood algorithms that are critical to success in certain cases.
    2. Automated molecular replacement program (Phaser)
    3. simultaneously uses all symmetry operators, resulting in a single peak with an improved signal-to-noise ratio which directly gives the position of the model in the unit cell. In addition, the TF is coupled with a PF to remove false maxima which correspond to interpenetrating molecules. Both the TF and PF allow the incorporation of a second model already placed in the cell. The TF solution may be subjected to rigid-body refinement incorporated in MOLREP. Non crystallographic symmetry may be imposed on the model in order to restrain the refinement. Pseudo-translation is automatically detected from analysis of the Patterson map. A significant off-origin peak gives the pseudo-translation vector, which is used to modify structure factors in the TF calculation (Navaza et al., 1998). In MOLREP multiple copies of the macromolecule in the unit cell can be searched (Vagin, 2000).
    4. MOLREP is an automated program for molecular replacement that utilizes a number of original approaches to rotational and translational search and data preparation. MOLREP can perform a variety of tasks that require rotational and/or positional search: standard MR, multi-copy search, fitting a model into electron density, heavy-atom search and model superposition. The arsenal of rotation (RF) and translation (TF) functions includes self-RF, cross-RF, locked cross-RF, phased RF, full-symmetry TF, phased TF, spherically averaged phased TF and packing function (PF).The program is general for all space groups. The output of the program is a PDB file with the atomic model ready for refinement and a text file with details of the calculations. The rotational search is performed using the RF of (Crowther, 1972), which utilizes the fast Fourier transform (FFT) technique. The default radius of the integration sphere is derived from the size of the search model and is usually two times larger than the radius of gyration. The RF solutions are refined prior to positional search using a rigid-body technique. The refinement is performed in space group PI and the outcome is evaluated by the correlation coefficient. It
    5. Automated molecular replacement program (MOLREP)
    6. include SORTING, that sorts, packs and assesses the quality of the experimentally measured diffraction data, and is run in the first step. The program TABLING calculates the continuous Fourier coefficients from the model placed in the artificial cell. The cross-rotation function is carried out by the program ROTING, which uses Crowther's algorithm (Crowther, 1972). TRAING is used to calculate the translation function. Finally FITING is used to refine the orientational and positional parameters of the molecule corresponding to the potential solutions, as a rigid body.
    7. To carry out MR, the AMoRe package can be used. AMoRe constitutes a suite of programs written by Jorge Navaza (Navaza, 1993; Navaza, 1994). These
    8. Automated molecular replacement package (AMoRe)
    9. was subsequently used as a probe model to carry out molecular replacement for one of the Fab-peptide complex; remainmg three Fab-peptide complexes were solved by using Ppy-LH as search model. The structure of antigen bound 36-65 Fab (2A61) was used for molecular replacement of two Fab-peptide complexes of the same antibody. AMoRe (Navaza, 1994) and Phaser packages from CCP4 suite (Elizabeth Potterton, 2003) were used for structure determination of antigen free BBE6.12H3 Fab and its complexes with peptide, respectively. The solution for 36-65 complexes was determined by using MOLREP from CCP4 suite. Both for MOLREP and AMoRe, calculations for rotation/translation functions were carried out using structure factors from 8 to 4 A resolutions. The transformation matrices obtained from AMoRe for antigen free Fab was utilized to orient the models in the corresponding unit cell. However, both Phaser and MOLREP have a module which automatically does orientation. The packing function of Phaser also checks for possible clashes or voids between the symmetry related molecules. All the solutions were unambiguous. For outputs of AMoRe and MOLREP the crystal packing was examined using Coot (Emsley P, 2004) to ascertain the absence of steric clashes or large voids between symmetry related molecules. Calculations of the Matthews coefficient (Kantardjieff and Rupp, 2003) indicated presence of two molecules for antigen free Fab and a single Fab molecule for all Fab-peptide complexes within the asymmetric unit.
    10. wavelength component in three dimensions inversely proportional to their values of h, k and /. The image of the object can be reconstructed by recombining the individual sine waves as occur in the objective lens of the microscope. Since it is not possible to focus the X-rays, only the intensities could be recorded with the loss of phases, well known as phase problem of crystallography. Macromolecular crystal structures are usually solved using one of the three techniques; multiple isomorphous replacement (MIR), multiple anomalous dispersion (MAD) or molecular replacement (MR). Of the three, MR is generally used in cases where a structural homolog is available. Since the structure of a number of antibodies is already known, MR is the method of choice for structure determination of antibody Fab. The molecular replacement method, involves orienting and positioning a model molecule in the experimental unit cell through rotations and translations. The rotation function developed by Rossman and Blow ( 1962), involves rotation of the Patterson function of one group or molecule with respect to the other in all possible ways and the ultimate superimposition of the two Patterson functions. The translation function deals with positioning the oriented molecule in the unit cell of the unknown structure. It utilizes the cross vectors between various symmetrically related molecules for positioning the probe in the target unit cell. The translation function is carried out by moving the oriented model in small increments along all three directions and calculating the correlation between observed and calculated intensities. From the solutions obtained, the one with the highest correlation and lowest R-factor was chosen for molecular replacement. The structure of the Fab of putative anti-NP germ line mAb Nl G9 was used for molecular replacement. The refined model of the native unliganded germline Fab
    11. The goal of diffraction analysis is reconstruction of the detailed structure of the asymmetric unit from a diffraction pattern. The diffraction pattern breaks down the structure into discrete sine waves. Any shape can be presented in three dimensions as the sum of sine waves of varying amplitudes and phases. The individual reflections of a diffraction pattern represent such waves, which have
    12. Structure determination using molecular replacement
    13. Structure determination
    1. Transformation of the bacterial host with an appropriate plasmid was performed using the method of Mandel and Higa ( 1970). A vial of competent bacterial cells was thawed on ice. The plasmid DNA was added at a concentration 1 ng/25 Jll of competent cells and the mixture was allowed to stand on ice for 30 min. The cells were given a heat shock by incubating the mixture at 42 °C for 90 sec, followed by a 2 min. incubation on ice. The mixture was diluted 10-fold with LB and incubated at 37 °C for 1 h. Afterwards the cells were plated on the LB-agar containing the antibiotic whose resistance marker was present in the plasmid.
    2. Transformation of Bacterial Host
    3. Oligonucleotides used in this study were synthesized by Rama Biotechnologies (Hyderabad, India).
    4. Oligonucleotides
    1. In addition, cryosections of an ovary from a normal cycling female (10 years) were also processed. Sections passing through a follicle were selected, washed in PBS and blocked for 30 min in 5% normal goat serum. The sections were incubated at 37°C with 1 :250 dilution of rabbit pre-immune and immune sera for 1 h, washed with PBS and incubated for 1 h with 1 :2000 dilution of goat anti-rabbit lg-FITC conjugate. Slides were washed with PBS and mounted in Glyceroi:PBS (9: 1) and examined under fluorescent microscope.
    2. A 3 year old monkey was treated daily for 3 days with an intramuscular injection of 25 IU of PergonaJ® (Laboratoires Serono S.A., Aubonne, Switzerland). The monkey was ovarectomized on day 6, and the ovary was snap frozen in liquid nitrogen and sections of 5 J..Lm thickness were cut in a cryostat at -20°C and fixed for 20 min in chilled methanol.
    3. Immunofluorescence on Bonnet Monkey Ovarian Sections
    4. The staining solution was aspirated and the plates left at 27°C 0/N. Plaques whicl appeared as clear zones, were identified, marked and verified under the microscope Plaques were picked up using a sterile 200 J.Ll tip and viruses were allowed to diffuse ou 0/N in 200 J.LI of CM to make the plaque pick stock virus.
    5. Sf9 cells ( 1.8x 1 o6) seeded in a 35 mm culture dish were infected in duplicate with 100 J!l of the serial dilutions (1 oO to w-2) of the transfection supernatant for I h. The viral inoculum was aspirated and 1.5 ml of the cooled agarose overlay (1.5% LMP agarose, 0.5X CM) was added to each dish and allowed to set. 1 ml of CM was added to each dish and the plates were incubated at 270C for 5 days. Medium was removed and cells were stained with 2 ml of staining solution (0.03 % neutral red in 10 mM PBS) for 1 h.
    6. Plaque Assay for Isolating Viruses
    7. Conditions for expression of r-bZP3 in SG 13009[pREP4] cells transformed with the pQE-bZP3 plasmid were standardized. Cells were grown till A6oo=0.7 and induced with different concentrations of IPTG (0.5, 1, 2, or 4 mM) for a constant time period (3h) or induced with a 0.5 mM IPTG for different time periods (0, 1, 2, 3 or 5 h). Cells were harvested and analyzed by SDS-PAGE and immunoblot as described above.
    8. Standardization of Expression Conditions
    9. mixed by inverting tubes. Following an incubation on ice for 5 min, 150 J.tl of ice cold potassium acetate solution (prepared by mixing 60 ml of 5 M potassium acetate, II.5 ml of glacial acetic acid and 28.5 ml of water) was added. The mixture was incubated on ice for 5 min and centrifuged at I2,000 g for 5 min at 4°C. The supernatant was decanted into a fresh tube and extracted once with an equal volume of phenol equilibrated with 10 mM Tris, pH 8 and 1 mM EDT A (TE) followed by extraction with chloroform:isoamyl alcohol (24: 1 ). DNA was precipitated by adding 2 volumes of chilled ethanol, contents mixed and tube incubated on ice for 30 min. The pellet collected after centrifugation at 12,000 g for 15 min was washed once with 70% alcohol, dried and resuspended in 50 J!l TE. To remove RNA contamination contents of the tube were treated with 20 J.tg/ml RNAase for I5 min at RT. DNA was checked and analyzed after restriction digestion by agarose gel electrophoresis.
    10. Colonies obtained after transformation were inoculated in 5 ml LB and grown 0/N in the presence of 100 Jlg/ml ampicillin (LB Amp). Next morning 1.5 ml of the culture was centrifuged for I min at I 0,000 rpm in a microfuge. The supernatant was discarded and the pellet was resuspended in 100 Jll of chilled GTE (50 mM Glucose, 25 mM Tris HCI and 10 mM EDT A). After an incubation at room temperature (RT) for 5 min, 200 Jll of freshly prepared alkaline SDS (0.2 N NaOH, 1% SDS) was added and the contents
    11. Small Scale Plasmid DNA Isolation
    1. containing 2. 2 M formaldehyde and 50 % V /V formamide. The samples were chilled on ice for 5 mins. and loading buffer added. A Taq I digest of phi X 174 DNA, filled-in wi~h Klenow polymerase using 32P-dCTP, was used as size marker for electrophoresis. The gels were run at <5 Vjcm.
    2. Total RNA was resolved in formaldehyde -agarose gels as described by Maniatis et al., ( 1982 ) • In general, the electrophoresis was performed using 1.2 ~ 0 agarose gels containing 2.2 M formaldehyde and 1 X running buffer 0.04 M rnorpholinopropanesulfonic acid -MOPS, pH 7.0; 0.01 M sodium acetate; 0.001 M EDTA ). RNA samples upto 20 ug in 5 ul ) were incubated at 55°c for 15 minutes in 5 X gel buffer
    3. Electrophoresis of RNA.
    4. lectrophoresed on 0.7 % -1.2 % agarose gels in TAE or TBE buffer. Choice of the percentage of agarose and the electrophoresis buffer system was made following the guidelines of Maniatis et al., ( 1982 ). In general, upto 1 kb fragments were resolved on 1.2 % agarose gels using TBE buffer. For most other purposes, TAE buffer was used. Agarose gel electrophoresis was carried out as described by Maniatis et al., ( 1982 ) . The run was stopped when the bromophenol blue dye migrated to within 1 em -1.5 em from the edge of ' the gel, except when the sample had fragments smaller than 500 bp, in which case the elctrophoresis was terminated at an earlier stage. The gel was immersed in water containing 0.5 ug I ml ethidium bromide, for 30 minutes, to stain the DNA. When detecting very low amounts of DNA, the staining was done for 60 minutes followed by destaining in 1 mM Mgso4 for one hour at room temperature. The DNA bands were visualised on a short wavelength UV transilluminator ( Fotodyne, Inc., USA and photographed with a Polaroid MP-4 camera using Polaroid type 667 film.
    5. DNA digested with restriction enzymes was
    6. For rapid electrophoretic analysis of plasmid DNA prepared by miniprep protocol, or to monitor the progress of digestion during various cloning procedures, the DNA was resolved on short agarose gels, taking less than one hour for the run. The electrophoresis was carried out in TAE buffer using 8 em long gels with a comb of teeth size 0.4 x 0.2 em. The width of the gel was variable, depending on the number of samples to be analysed. Gels were run at 50 100 volts, till the bromophenol blue dye migrated to within 0.5 em of the edge of the gel.
    7. Mini gel electrophoresis
    8. Electrophoresis of DNA
    9. Agarose ~ electrophoresis
    10. Ampicillin, tetracycline, chloramphenicol and gentamycin were from Sigma. Geniticin ( G418 ) was from Gibco Laboratories, USA.
    11. Antibiotics.
    1. The membranes were suspended (1.4 x 108 cell equivalent) in 250 III of incorporation buffer (50 mM HEPES, pH = 7.4, 25 mM KCI, 5 mM MgCb, 5 mM MnCI2, 0.1 mM TlCK, 1 Ilg/ml leupeptin, 1 mM ATP, 0.5 mM dithiothreitol and 0.4 Ilg/ml tunicamycin). Each assay tube was prepared by adding 12.5 III of 1 % Chaps, 2.8 III of 200 IlM GOP-Man, 10 III of GOP-[3H]-Man (1IlCi) and 25 nmol of synthetic substrate (49). The contents were lyophilized and 250 III of membrane suspension (1 .4 x 108 cell equivalent in incorporation buffer) were added to each tube. The tubes were incubated at 28°C for 20 minutes, cooled to 0 °C and the membranes were pelleted at 4 °C for 10 minutes in a microcentrifuge. The eH] mannosylated products, that were recovered in the supernatant, were mixed with 0.5 ml 100 mM ammonium acetate and applied to a C18 Sep-pak cartridge that had been washed with 5 ml 80% propan-1-01 and 5 ml 100 mM ammonium acetate. The cartridge was washed with 1.5 ml of 100 mM ammonium acetate and then the eluate was reapplied to the same cartridge. The cartridge was subsequently washed with 5 ml of 100 mM ammonium acetate, after which the bound material was eluted with 5 ml of 60% propan-1-01. The final eluate was concentrated and redissolved in 100 III of 60% propan-1-01. One tenth of this volume was taken for scintillation counting. The above assay was then carried out with a range of concentrations of OMJ to assess it's effect on the activity of eMPT enzyme parse.
    2. eMPT inhibition assay
    3. mixture). These samples were lyophilized and 125 III of the reaction mixture was added to each tube. The tubes were then incubated at 25°C for 1 h and the biosynthetic LPG was extracted as described above. 10 III of the solvent E extract was taken for scintillation counting.
    4. 1. Mild acid hydrolysis: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and then suspended in 0.02 N HGI (200 Ill). The mixture was then placed in a 100 °G water bath for 5 minutes. After hydrolysis, the sample was again dried under nitrogen and codried thrice with toluene (0.5 ml). The residue was suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. 2. Nitrous acid deamination: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and then suspended in 0.2 ml of 0.125 M sodium acetate (pH = 4.0) and 0.25 M sodium nitrite. The mixture was incubated at 25 °G for 40 h. The sample was dried under nitrogen, suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. 3. PI-PLC treatment: 0.6 ml of the pooled solvent E soluble fractions was dried with a stream of nitrogen and suspended in 0.4 ml of PI-PlG buffer (0.1 M Tris chloride, pH = 7.4 with 0.1 % deoxycholate) and 0.2 ml of PI-PlG concentrate (B.subtifis culture supernatant) was added. The mixture was then incubated at 37 °G for 16 h. The sample was dried under nitrogen, suspended in 0.6 ml of 0.1 M NaGI in 0.1 M glacial acetic acid, loaded onto phenyl sepharose column and elution done in the same manner as described before. Fractions of 0.6 ml each were collected and assayed for radioactivity. The effect of deoxymannojirimycin (Sigma, Gat. no. 0-9160) on the cell free biosynthesis was carried out. OMJ (5 mg) was dissolved in 1 ml of MQ water and 2.5, 5, 25 and 50 III were transferred to eppendorf tubes separately (which corresponded to 0.5, 1, 5 and 10 IlM concentrations of OMJ in 125 III ofthe reaction
    5. Characterization of biosynthetic LPG
    6. NaGI in 0.1 M glacial acetic acid, 1.2 ml of 0.1 M glacial acetic acid, 0.6 ml of water and 3.6 ml of solvent E. Fractions of 0.6 ml each were collected and assayed for radioactivity.
    7. Parasites (6 X 109) were harvested, pelleted at 3000 g for 10 min, washed with PBS, repelleted and suspended in 10 mL of HEPES buffer (100 mM HEPES-NaOH, pH = 7.4, 50 mM KCI, 10 mM MnCI2, 10 mM MgCI2, 0.1 mM TLCK, 1 Jlg/mL leupeptin) containing 10% glycerol. The cells were disrupted in a Parr nitrogen cavitation bomb (1500 psi, 25 min, 4°C, 3 cycles). The debris was removed by centrifugation at 3000 g for 5 min and the supernatant was centrifuged at 100,000 g for 1 h at 4°C. The resulting membrane pellet was resuspended in 10 mL of HEPES buffer without glycerol and centrifuged again at 100,000 g for 1 h at 4°C. The membranes were finally suspended in 1 mL (13 mg/mL) of HEPES buffer without glycerol. The incubation mixture per reaction contained membrane protein (2 mg) in 125 JlL of 50 mM HEPES-NaOH buffer, pH = 7.2 containing supplements (25 mM KCI, 5 mM MgCI2, 5 mM MnCI2, 0.1 mM TLCK, 1 JlglmL leupeptin, 0.8 mM ATP, 0.4 mM On) with 2 JlM UOP-[3H]-galactose (2 JlCi) and 10 JlM GOP-mannose. The mixture was incubated at 25°C for 1 h, terminated by the addition of CHCI~CH30H (3:2) to give a final ratio of CHCI~CH30H/H20 (3:2:1) and sonicated. The layers were then allowed to separate out after which the lower layer was removed with the aid of a micropipette. The tube containing the upper and intermediate layer was centrifuged (10,000 rpm, 4°C, 5 minutes). The supernatant was discarded and the resultant pellet (membranes) was suspended in 1 mL of CHCI~CH30H/H20 (1:1 :0.3). The solution was again centrifuged (10,000 rpm, 4°C, 5 minutes) and the pellet was extracted with 1 mL of solvent E (H20/ethanol/diethylether/pyridine/NH40H 15:15:5:1 :0.017) thrice. The solvent E extracts were pooled and dried under a stream of nitrogen, suspended in 0.6 mL of 0.1 M NaCI in 0.1 M glacial acetic acid and chromatographed over a 1 mL column of phenyl sepharose. Phenyl Sepharose Column of Biosynthetic LPG. The solvent E extract suspended in 0.6 mL of 0.1 M NaCI in 0.1 M glacial acetic acid was applied to a column (0.5 x 2 cm) of phenyl sepharose (Pharmacia BioteCh), preequilibrated with 0.1 M NaCI in 0.1 M glacial acetic acid. The column was then washed sequentially with 3 mL of 0.1 M
    8. Cell-free Biosynthesis42 of LPG using Leishmania membranes
    9. Inhibition of LPG biosynthesis: Identification of deoxymanno-jirimycin (DMJ) as an inhibitor that targets elongating MPT enzyme
    1. incubations were carried out for I h at RT and each incubation was followed by three washings with PBS containing 0.1% Tween-20 (PBST). Post-blocking, the membranes were incubated with 1:1000 dilution ofMA-813 ascites (for detection ofr-bmZP1), MA-451 ascites (for detection of r-dZP3) or rabbit polyclonal anti-r-rG antibodies (for detection of r-rG), followed by an incubation with 1:5000 dilution of goat anti-mouse or goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase (HRPO) (Pierce) respectively. The blots were developed with 0.6% (w/v) 4-chloro-1-naphthol in 50 mM PBS containing 25% methanol and 0.06% H202• The reaction was stopped by extensive washing with double distilled water
    2. The cells (2 - 4 x 1 06) transfected with plasmid DNA were resuspended in minimum volume of 2X sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% P-mercaptoethanol, and 0.001% bromophenol blue). The samples were boiled for 10 min and resolved on a 0.1% SDS-1 0% PAGE (Laemmli, 1970). The expression of recombinant proteins was analyzed by Western Blot. The proteins were electrophoretically transferred to 0.45 J.lm nitrocellulose membrane 0/N at a constant current of 30 rnA (milliampere) in Tris-Giycine buffer (25 mM of Tris-HCl and 200 mM glycine) containing 20% methanol (Towbin et al., 1979). Post-transfer, the membranes were washed once with PBS and non-specific sites were blocked with 3% BSA in PBS for 90 min at RT. All the subsequent
    3. Analysis of expressed recombinant protein by immunoblot
    4. The PCR products obtained by amplification were resolved on a 0.8% low melting point (LMP) agarose gel using IX TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDT A) and purified from the gel. The purified PCR products were first blunt-ended at 72°C for 30 min using 0.5 units (U) of cloned Pfu polymerase, 1 OmM dNTPs, 1 OX polishing buffer (Stratagene). These PCR products were ligated separately to pPCR-Script Amp SK ( +) cloning vector, using vector to insert ratio of 1 :20 in a 10 Jll reaction volume for 3 h at room temperature (RT). The reaction mixture contained 10 ng of pPCR-Script Amp SK(+) cloning vector, 4 U ofT4 DNA ligase, 0.5 Jll of 10 mM rATP, 1 Jll of lOX reaction buffer, 5 U of S1f I restriction enzyme. The buffers and enzymes used were supplied along with the PCR-Script™ Amp cloning kit (Stratagene). For dZP3-rG fusion, the PCR amplified product was ligated with pGEM-T Easy vector (Promega) without blunting. The reaction mixture contained 50 ng pGEM-T Easy vector, 130 ng of fusion PCR product, 3 U ofT4 DNA ligase and 5 fll of2X Rapid Ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgC}z, 10 mM DTT, 2 mM ATP and 10% polyethylene glycol). The reaction was carried out at 16°C for 16 h.
    5. Agarose gel electrophoresis and ligation of PCR amplified fragments in pPCR-Script Amp SK (+)cloning vector
    1. Theenzyme-linkedimmunospot(ELISPOT)assaywasusedfortheenumeration of totalimmunoglobulinsecretingcells(ISC)andantigen(BCG)-specificantibody-secretingcells(ASC)inthespleenandtheblood.TheELISPOTassayfollowedby Aaltonenetal.(1994)wasused.
    2. EnumerationofsecretinglymphocyteswithELISPOTassay
    3. LDHisthekeyenzymeinvolvedinglycolysis,andisresponsiblefortheanaerobicconversionofpyruvicacidtolacticacid,theterminalstageintheEmbden-Meyerhofpathway.Theenzymeactivitywasdeterminedinthecontrolandeffluentexposedfishbrain,gill,muscle,liver,heart,kidneyandair-breathingorgansfollowingSrikantanandKrishnamurthi(1955).TheopticaldensitiesweremeasuredinaUVSpectrophotometerusing340 nmfilterandtheresultsare expressedaspmolesofformazanmg'1proteinhr’1
    4. Lactatedehydrogenase(LDH)(L-LactateNADoxidoreductase)(EC1.1.1.27)
    5. micro-haematocrittubewasfilledto100mmwithanticoagulatedblood.Oneendofthetubewassealedwithsealingwaxandthetubewasthenkeptinaverticalpositioninaglassbeakerstuffedwithcotton.Afteronehour,lengthoftheplasmacolumnwasmeasuredwitha rulergraduatedin0.5mm.
    6. ESRwasdeterminedbythemicromethodbecausethequantityofbloodavailablefromindividualfishwasinsufficienttoadoptanymacromethod.Anon-heparinised
    7. ErythrocytesedimentationRate(ESR)
    8. ThecircadianrhythmofbimodalO2uptakeofcontrolandeffluenttreatedfisheswerestudiedseparatelyat28°±1°C.TheamountsofO2extractedfromwaterandairwereseparatelydeterminedforadayatregularintervalsof3hreach.TotalO2uptakeateachtimewasobtainedbysummingupthevaluesforaquaticandaerialrespirationobtainedatthecorrespondingtime.Throughoutthepresentstudy,theinitialO2contentofthewaterwaskeptconstant(6±0.5mgF1)
    9. CircadianrhythmofbimodalO2uptake
    10. experiments.Chemicalcharacterizationoftheeffluent(Table1)wascarriedoutbyusingstandardmethods(APHA,1985)
    11. ThecementfactoryeffluentobtainedfromtheACCcementfactory,Madukkarai(Figure6)in101blackpolyethylenecontainerswerekeptinarefrigeratoruntilusedfor
    12. EffluentCollection
    1. with 4 ml of equilibration buffer QBT (750mM NaCl, 50mM MOPS, pH 7.0, 15% isopropanol, 0.15% Triton X-100) and the column was allowed to empty by gravity flow. The supernatant was applied to the QIAGEN-tip and allowed to enter the resin by gravity flow. The QIAGEN-tip was washed thrice with 10ml of wash buffer QC (l.OM NaCl, 50mM MOPS, pH 7.0, 15% isopropanol). The DNA was then eluted with 5 ml of elution buffer QF (1.25M NaCl, 50mM Tris-Cl, pH 8.5, 15% isopropanol). The DNA was precipitated by adding 0.7 volumes of isopropanol to the eluted DNA. It was thoroughly mixed and centrifuged immediately at 13,000 rpm for 30 min at 40C. The supernatant was carefully decanted. The DNA pellet was washed with 2 ml of 70% ethanol, and centrifuged at 13,000 rpm for 15 min at 40C. The supernatant was carefully decanted without disturbing the pellet. The pellet was air dried for 5-10 min and the DNA was dissolved in 200 p.l of RNase-DNase free water. To determine the yield, DNA concentration was determined both by Ultra Violet (UV) Spectrophotometry (DU-65 spectrophotometer, Beckman, U.S.A.) and quantitative analysis on an agarose gel using a UV Transilluminator (UVP, California, U.S.A.). All the putative clones were then screened for the correct recombinant clones by restriction enzyme digestion using appropriate enzymes. The digested samples were checked on an agarose gel along with an appropriate size marker to assess the size of the insert from the putative clones. The clones containing very small fragments were further confirmed by sequencing both strands of the DNA
    2. For large scale plasmid DNA isolation, the bacterial cells were cultured in 100ml of LB medium with 100pg/ml of ampicillin. The cultures were grown for 8-10 hours at 37<>C with vigorous shaking (-200 rpm). Plasmid DNA was isolated using the QIAGEN Plasmid Midi kit (100). Briefly, the bacterial cells were harvested by centrifuging at 6000 rpm for 10 min at 4<>C. The bacterial pellet was resuspended in 4 ml of the resuspension buffer P1 (50mM Tris-Cl, pH 8.0, 10mM EDTA, 100l!g/ml RNase A). 4 ml of lysis buffer P2 (200mM NaOH, 1% SDS) was added, mixed gently by inverting 4-6 times and incubated at room temperature for not more than 5 min. Further 4 ml of chilled neutralization buffer P3 (3.0 M potassium acetate, pHS.S) was added, mixed gently as before and incubated on ice for 10 min. It was then centrifuged at maximum rpm for 30 min at 4<>C. The supernatant containing the plasmid DNA was immediately removed andre-centrifuged at 10,000 rpm for 15 min at 4<>C. The supernatant was now collected in fresh tubes and kept on ice. A QIAGEN-tip 100 was equilibrated
    3. lasmid Midipreps:
    4. DNA restriction enzymes were purchased from New England Biolabs (Massachusetts, USA) and Promega Corporation, (Madison, U.S.A.). RNase A was obtained from Qiagen (West Sussex, U.K.). DNA ligase, RNA polymerase, RNAsin, Taq DNA polymerase, T7 RNA polymerase, SP6 RNA Polymerase and alkaline phosphatase were obtained from Promega Biotech.
    5. Enzymes
    1. Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed, from 35mm dish and 1ml to TRIzol was added directly to the dish and kept at room temperature for 5 minutes. The cells were harvested by pipetting up and down three four times and transferred to a 1.5ml microfuge tube. For each 1mlTRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed for 10 minutes. The upper aqueous phase was transferred into a fresh microcentrifuge tube and 500 μl of isopropanol was added and this was incubated at -20°C for 1 hour. The RNA was pelleted by centrifugation at maximum speed for 30 minutes at 4°C. The supernatant was decanted and the pellet washed with 1ml of 70% ethanol followed by a second wash with 1ml of 90% ethanol and centrifugation at maximum speed for 10 minutes. The supernatant was removed and the pellet air-dried for about 5 minutes and re-solubilized in 30-50 μl RNase free deionized (DEPC-treated Milli-Q) water and aliquots were stored at -70°C
    2. Total RNA isolation from cultured mammalian cells
    1. 5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine
    2. Activation of SEND arrays for peptide analysis
    3. DNA fragments to be used for ligation were eluted from the agarose gel, after the electrophoresis. The gel piece containing the desired band of DNA was sliced out and the DNA was purified from the gel using the purification kits available for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel
    4. Purification of DNA fragments by elution from the gel
    5. Primers used in this study
    1. The growth pattern of wild-type and transfected cells were carried out counting the number of cell in a haemocytometer at 24hr intervals. For this equal number of cells (107 or 5 X106) of wild-type and transfected parasites in log phase were plated. An aliquot was withdrawn every 24 hours and used for counting in a counting chamber to analyse growth pattern.
    2. Growth curve analysis
    3. get a linear amplification of serial dilutions of e-DNA. Equal quantities of RNA (2~-tg) were used for e-DNA preparation for control and treated samples. Then identical PCR reactions were set for all experimental groups, according to the primer pair. Equal volume of PCR products were visualized by agarose gel electrophoresis as described below. The bands were quantified by densitometry using Labworks™ Image Acquisition and Analysis Software (UVP Bioimaging Systems, UVP Inc., Upland, CA)
    4. Relative expression levels of specific gene(s) in treated cells were determined by semi-quantitative PCRs. Initially standardization was carried out for each primer pair (Table 3.5) to determine cycle number to
    5. Semi-quantitative RT -PCR
    6. Syrian hamsters (Mesocricetus auratus), 3-6 weeks old, were used as in vivo Leishmania infection models. The clinicopathological features of the hamster model of VL closely mimic active human disease. Promastigotes in the stationary phase were harvested at 1258 x g for 10 min at RT and washed several times with sterile PBS to remove all traces of medium and FBS. They were resuspended to a cell count of 2X109 cells/mL in PBS. 10011L of this (108 parasites) were injected intra-cardially into hamsters and infection allowed to proceed for 2 months. After 2 months, the hamsters were euthanized with C02 and the spleens were harvested. The spleens were first weighed, and then cut transversely. The exposed surface was gently pressed onto a clean slide to make imprints which were allowed to air dry. Following this, the smears were fixed either in chilled methanol for 5 min or 4% formaldehyde for 10 min. The slides were washed with PBS and either stored in -70°C for later use or stained with Giemsa stain to visualize infection
    7. In vivo infection of Syrian hamsters with Leishmania donovani parasites
    1. Oligonucleotides and PCR products were end labeled using phage T4-polynucleotidekinase (PNK, New England Biolabs) with 32P-γ-ATP. The radiolabelling reactionmixture (50 μl) contained 1 X of buffer provided by the company, 10 units of T4-PNKand 50 μCi of32P-γ-ATP. The reaction mix was incubated for 1-hr at 37ºC and thereaction was stopped by adding 10 μl of 0.5 M EDTA. The labeled oligonucleotides andDNA fragments were purifiedeither by the Qiagen PCR purification or nucleotide removal kit.Labelling efficiency was checked by scintillation counting
    2. Radiolabelling of oligonucleotides and PCR products
    3. Native isoelectric focusing was done using Pharmacia Phast Gel Apparatus and precast IEF gel (pH 3-9) from GE healthcare. The samples were prepared in 50 mM sodium buffer (pH 8.0) and applied in the middle portion of the gel. Gels were run as previously described(Olsson et al., 1988) that is at 15°C, pre-focusing at 2000 V (75Vh), sample loading at 200V (15Vh) and run at 2000V (500Vh). Staining was done using Coomassie Blue G-250
    4. Native Isoelectric Focusing
    5. DNA fragments to be used for specific purposes like ligation or radioactive labeling were eluted from the agarose gel after electrophoresis. The gel piece containing thedesired band was sliced out from the gel and the DNA was purified using commerciallyavailable purification kits for this purpose. The efficiency of elution was determined bychecking a small aliquot of DNA sample on the gel
    6. Purification of DNA by gel elution
    7. supplemented with amino acids and appropriate antibiotic and grown at 37ºC to an A600of 0.5-0.6. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysedwith addition of one drop of chloroform and 1-2 drops of 1% SDS solution. 0.2 ml offreshly prepared 4 mg/ml ONPG was added to start the reaction and incubated at roomtemperature till the color of the reaction mixture turned yellow. 0.5 ml of 1 M Na2CO3was added to stop the reaction and the time duration from initial addition of ONPG tothe stopping of reaction was noted.The absorbance of reaction mix was taken at 420nm and 550 nm. The A600of the culture used was also noted. The enzyme specificactivity (in Miller units) was calculated using following equation:β-galactosidase specific activity = [1000 X A420-(1.75 X A550)] / t X v X A600Where t isthe time period in minsand v the volume of culture used in ml.Each value reported is the average of at least three independent experiments, and the standard error was <10% ofthe mean in all cases
    8. β-galactosidase assay was performed according to Miller (1992). An overnight grownculture of the bacterial strain was sub-cultured in glucose Minimal A medium
    9. β-galactosidase assay
    10. Thialysine or thiosine (S-Aminoethyl-L-cysteine)is a toxic analog of Lys. Strains were testedfor sensitivity/resistance to thialysine by streaking them on minimal A-glucose platessupplemented without and with100-200 μg/ml thialysine(Steffes et al., 1992)
    11. Test for thialysine resistance
    12. Waterto 3 mlTEMED 10 μlDenaturing (urea) sequencing gel (6%) composition10 X TBE 50 ml40% acrylamide 75 mlUrea 210 gm (7 M)Waterto 500 mlThis was filtered through a 0.45/0.22 μ milipore filter.For casting the gel 35 ml of the sequencing gel mixure was mixed with 150 μl10% APS and 25 μlTEMED
    13. Formaldehyde agarose gel(For 50 ml)DEPC treated water 43 mlMOPS buffer 5.3 mlAgarose0.63 gmFormaldehyde2.6 mlThe above mix was boiled without formaldehyde to dissolve agarose and then at around 50ºC formaldehyde was added just before casting the gel.40% Acrylamide solutionAcrylamide39 gmBis-acrylamide 1 gmWater to 100 mlNon denaturing gel composition (50 ml)40% acrylamide solution 5 ml10 X TBE 5 mlH2O 40 ml10% APS 250 μlTEMED 30 μlSDS PAGE gel (12%)For resolving Gel (15 ml):30% Acrylamide solution 6 ml1.5 M Tris-Cl (pH 8.8)3.8 ml10% SDS150 μl10% APS 150μlWaterto 15 mlTEMED 10 μlFor stacking gel (3 ml):30% Acrylamide solution 500 μl1 M Tris Cl (pH 6.8) 380 μl10% SDS 30 μl10% APS 30 μl
    14. Storage buffer for proteinTris-Cl (pH 8.0) 20 mMNaCl 300 mMDTT10 mMGlycerol 40 % Hybridization bufferTris-Cl (pH 8.0) 9 mMEDTA 0.35 mMSample buffer (for SDS-PAGE)Tris-Cl (pH 6.8) 150 mMSDS (20%) 6% v/vGlycerol 30% v/vβ-mercaptoethanol (5%) 15%Bromophenol blue 0.6% (w/v)EMSA binding bufferTris-Cl (pH 7.5) 10 mMNaCl 50 mMEDTA1 mMGlycerol 5 %DTT 5 mMDenaturing gel loading buffer with dyeFormamide 95%EDTA 20 mMXylene Cyanol 0.05 gmBromophenol blue0.05 gmNon denaturing gel loading buffer with dyeTris-Cl (pH 7.5) 250 mMBromophenol blue 0.02%Glycerol 20%
    15. MOPS bufferMOPS 4.16 gm0.5 M EDTA 1.0 mlSodium acetate 0.68 gmWater (nuclease free) to 500 mlIt was filter sterilized and stored in an amber colored bottle. This was prepared as 10 Xstock solution and used at 1 X concentration.INOUE (PIPES) bufferPIPES (free acid) 10 mMCaCl2.2H2O15 mMKCl 250 mMMnCl2.4H2O 55 mMpH was adjusted to 6.7 with 1 N KOH.PIPES gets into solution when the pH is greater than 6.7. MnCl2was dissolvedseparately and added drop by drop with stirring. The pH was adjusted to 6.7 and filtersterilized and stored at –20ºC.Z buffer (for β-Galactosidase assay)Na2HPO416.1 gmNaH2PO45.5 gmKCl0.75 gmMgSO4.7H2O 0.246 gmβ-mercaptoethanol 2.7 mlWaterto 1000 mlpH was adjusted to 7.0 and stored at 4ºC.SDS running bufferTris-base 30.3 gmGlycine144 gmSDS 10 gmWaterto 1000 mlIt was prepared in 10 X concentration and diluted to 1 X for running
    16. Citrate bufferCitric acid (0.1 M)4.7 volumeSodium citrate (0.1 M) 15.4 volumeTE bufferTris-Cl (pH 8.0) 10 mMEDTA 1 mMTBE bufferTris-Borate 90 mMTris-Borate 90 mMEDTA (pH 8.0) 2 mMThis was prepared as 10 X stock solution and used at 1 X concentration.TAE bufferTris-acetate 40 mMEDTA (pH 8.0) 2 mMThis was prepared at 50 X concentrated stock solution. Both TBE and TAE were usedas standard electrophoresis buffers
    17. Buffers and solutions
    1. For determiningthe intracellular pH from fluorescence intensity values of CFDA-SE-loaded cells, anin vivocalibration curve was prepared between fluorescent intensity and pre-adjusted environmental pH values. Briefly,CFDA-SE-loaded wild-typeC. glabratacellswere incubatedwith 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma# C2759) at 30 ̊C for 10 min in 50 mM CP buffer adjusted to different pH values ranging from 4.0 to 7.5, with an interval of 0.5 unit.CCCP is an ionophore which dissipates the plasma membrane pH gradient, thus, rendering the intracellular pH similar to the extracellular pH. Fluorescent intensities were determinedand a calibration curvewas plotted between the ratio of intensity at 490 to430 nm versuspH.A polynomial distribution of fluorescent intensity signal and pH was observed for CFDA-SE probe(Figure2.1)and the graphequation was used todeterminethe intracellular pHof C. glabratacells
    2. In vivointracellular pH calibration curve
    3. and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C
    4. C. glabrataCgYPS7ORFwas cloned in a self-replicating pGRB2.2plasmidwhich contains C. glabrata CEN-ARS, S. cerevisiaeURA3gene, S. cerevisiaePGK1promoter and C. glabrataHIS3-3′ untranslated region. For cloning CgYPS7in pGRB2.2,CgYPS7ORF (1.764 kb) was PCR-amplified from the wild-type genomic DNA with high fidelity Platinum PfxDNA polymeraseusing primers carrying restriction sites for XbaIand XhoI. The1.764 kb amplifiedPCR product waspurified with QIAquick PCR purification kit (Qiagen # 28104),digested with XbaI and XhoI and cloned in the pGRB2.2plasmid at XbaI–XhoI sites in the multiple cloning site (MCS)region downstream of the PGK1promoter.Positiveclones were verified by PCR, sequencing and complementation analysesofCgyps7∆mutant. Yeast transformantsobtained by lithiumacetate methodwere selected on plates lacking uracil
    5. Cloning of CgYPS7gene
    6. All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis
    7. Cultivation of logarithmic-phase cell culture
    8. Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially
    9. synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study
    10. Oligonucleotides
    11. 10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use
    12. Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)
    13. Other buffers
  2. shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
    1. was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula
    2. Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel
    3. Adherence assay
    4. After 2-3 h exposure,phosphorimagerscreenwas scannedon Fuji FLA-9000 to acquire hybridization images. Next,signal intensity for each spot on the membrane for both input and outputsampleswas quantifiedusing Fuji Multi Gauge V3.0 software andpercentage intensity foreach spot relative tothe whole signal intensity ofthe membranewas determined.To identify mutants with altered survival profiles,ratio of output (Op) to input (Ip) signal for each spot (oligonucleotide tag)present on the membranewas calculated.Mutantsdisplaying at least 6-fold higher and 10-fold lower survival were selectedas “up’ (Op/Ip= 6.0) and ‘down’ (Op/Ip = 0.1) mutants, respectively
    5. Data analysis
    6. Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days
    7. Yeast transformation
    8. Forinfection of THP-1 cells with single C. glabratastrain, PMA-treatedTHP-1 monocytes were seeded in 24 wellcell culture plate toa seeding density of 1 million cells per well. To prepare C. glabratacells for macrophage infection, single colony of the desiredstrain wasinoculated in YPD medium and allowed to grow for 14-16 hat 30°C. C. glabratacellsfrom 1ml overnight culture were harvested, washed with PBS andcell density was adjustedto 2X107cells/ml.50 μl of thisC. glabratacell suspension wasinfectedto macrophages to a MOIof 10:1. Two hours post infection, infected THP-1 macrophages were washed thrice with PBS to removenon-phagocytosed yeast cells and medium was replacedwith fresh prewarmed medium. Atdifferent time points post infection,infected THP-1 macrophages were washed with PBS three timesandlysed in 1 mlsterilewater. Lysates were collected by scrapping the wells with a micropipette tip, diluted in PBS and appropriatelysatedilutions were platedon YPD agar medium. Plates wereincubated at 30°C and colony forming units (CFU) were counted after 1-2 days. Final CFUs per ml were determined by
    9. multiplying CFUs with dilution factor and fold-replication was determined by dividing the CFUs obtained at 24 h time-point by 2 h CFUs
    10. Single infection assay
    11. 10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4LB-ampicillin and LB-kanamycin platesLBmedium50 μg/ml ampicillin30 μg/ml kanamycinMedia and solutions were sterilizedeither by routine autoclaving at 121°C and 15 psi for 20 minor by filtration through membrane of 0.22 μm porosity
    12. Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone
    13. Bacterial media
    14. Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replaced withother carbon sourcesviz.,sodium acetate, ethanol, oleic acid, glycerol and citric acid.Yeast Nitrogen Base (YNB) without ammonium sulphate and amino acids0.17% Yeast Nitrogen Base2% DextroseCasamino Acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acidsFor preparing plates, 2% agar was added tothe medium before autoclaving
    15. Yeast media
    16. Media
    1. Of theligation mixture,2μl (of total volume of 10 μl reaction)was added to atube of 100μlultra competent DH5α bacterial cells and incubated in ice for 30 minutes. The tubewas quickly transferred to a water bath maintained at 42°C to give a heat shock for 90 seconds and again quickly transferred to ice. 1ml of LB broth was added to the tube and then incubated at 37°C for 1 hour. The bacterial cells were then pelletdownby centrifugation at 6000 rpm for 5 minutes and plated on LB agarcontaining appropriate antibiotic
    2. Transformation of ligated DNA
    3. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions
    4. DNA sequencing
    5. BCA (Bicinchoninic acid) method was used to determine the proteinconcentrationin various samples. The Cu2+ions from cupric sulphate (present inBCA reagent B) reagent arereduced to Cu+by the protein in an alkaline medium. The cuprous ion (Cu+) then combines with BCA (present in BCA reagent A) to give a purple colour whose intensity is proportional to the amount of protein present in the samples. This intensity is measuredby colorimetry at 562 nm. BCA reagent was prepared by mixing reagent A with reagent B in avolumeratio of 50:1. A standard curve was generated using increasing concentrations of BSA (2-10μg) in a 25μl reaction, in a 96 well plate. Cell lysates were also dilutedto same volume in parallel wells. 200μl of BCA reagent was then added to each well and incubated at 370C for 30 minutes. The absorbance readings were then takenin a spectrophotometer at 562 nm. Total protein was quantified by calculation of the slopes of regression lines ofabsorbanceand BSA standards
    6. Protein estimation
    7. Nuclear extractionbuffer (without protease inhibitors)
    8. Cytoplasmic extractionbuffer (without protease inhibitors)
    9. For Cell fractionation
    10. Transfer Buffer
    11. Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH
    1. The method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve
    2. Cell proliferation assay
    3. 6X DNA loading dye
    4. Agarose gel
    5. TAE
    6. For DNA electrophoresis
    7. (e) Stacking polyacrylamide gel
    1. growntill the OD600reached 0.8-1.0. Cells equivalent to 1OD600of each culture was taken for the labellingof total protein. Cells were washed in methionine-free synthetic complete medium(SC-Met) twice, suspended in SC-Met mediumcontaining 25μCi/mLof 35S Met-Cys twin label mixand incubatedfor 1min, 5 min and 15min. Cells were washed twice in ice-cold SC-Met medium twice and suspended in 500 μL of Tris-salinecontaining protease inhibitor cocktail.To this,300 μL ofglass beads (0.45-0.6mm diameter)were added and cells were lysed for 10 min by bead beating (with intervals of 1 min on time and 30 sec off time).The lysate was centrifuged at high speed for 15 minat 4°C. To the supernatant,sodium deoxycholate was added to a final concentration of 0.1 mg/mLand incubated on ice for 30 min. To this solution, 20% trichloroacetic acid was addedto a final concentration of 6%, incubated for 1 h on ice, and centrifugedat high speedfor 20minat 4°C. The pellet was suspended in 300 μL of Tris-saline and counted inaliquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5
    2. Wild type and knock out yeast strains were grown in YPD (Difco) whereas synthetic complete medium without uracil was used for the KCS1 complementedstrains. Overnight grown yeast were subcultured in appropriate medium at 0.2 OD600and
    3. Protein synthesis analysis
    4. Wash buffer II10 mM Tris-HCI,pH 8.01 mM EDTA250 mM LiCl0.75% NP-400.75% sodium deoxycholateProtease inhibitor cocktailElutionbuffer II50 mM Tris-HCl,pH 8.0 10 mM EDTA 1% SDS
    5. Lysis buffer50 mM HEPES,pH 7.5140 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail (added fresh)Wash buffer I50 mM HEPES,pH 7.5500 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail
    6. Buffers for chromatin immunoprecipitation
    7. 0.5% Yeast Extract 1% Tryptone 1% NaClLB-ampicillin plates LB medium 100 μg/mL ampicillin Media and solutions were sterilized either by routine autoclaving at 121°C and 15 psi for 20 min or by filtration through membrane of 0.22 μm porosity.For yeast and bacterial growth, plates were preparedby adding 2% to the medium before autoclaving

      Italic

    8. Luria-Bertani (LB) medium forbacterialgrowth
    9. Yeast synthetic complete medium without leucine(SC-Leu)0.67% Yeast Nitrogen Base without amino acids 76mg/L His76mg/L Ura76 mg/mL Trp76 mg/mL Met2% DextroseYeast sporulating medium1% Potassium acetate0.05% Dextrose
    10. Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone 2% Dextrose Yeast synthetic complete medium(SC)0.67% Yeast Nitrogen Base with amino acids 2% Dextrose1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil76 mg/L uracilYeast synthetic complete medium without histidine(SC-His)0.67% Yeast Nitrogen Base without amino acids 1.92 g/L Yeast Synthetic Drop-Out media supplement without histidine2% DextroseYeast synthetic complete medium without uracil(SC-Ura)0.67% Yeast Nitrogen Base without amino acids 1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil2% DextroseYeast synthetic complete medium without methionine(SC-Met)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76 mg/L His76mg/L Ura2% DextroseYeast synthetic complete medium without tryptophan(SC-Trp)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76mg/L His76mg/L Ura76 mg/L Met2% Dextrose
    11. Yeast media(Media composition was followed as described by Sigma product data sheet)
    12. Media
    1. The plasmid-DNA/PEI mixture was incubated for 15 minutesat room temperature.The mixture was added to cells,andmixed properlyby rocking the culture plate back and forth. Cells were incubated at 37°C in a CO2 incubator.The transfected cells were harvested at 24-48 hours post-transfection
    2. Cells were plated inthe cell culture dishes one day before transfection in RPMI1640 supplemented with FBS and penstrep (complete medium). All the reagents were brought to room temperature before starting transfection. Plasmid-DNA was diluted in serum-free medium and PEI was added(Table 9)Table 9: PEI plasmid-transfection methodology
    3. Plasmid transfection using PEI
    4. Table 8: Lipofectamine plasmid-transfection methodology
    5. For transfection with Lipofectamine, cells were plated in antibiotic-free medium 24 h before transfection and were transfected at a confluency of 70-80% as per the manufacturer’s protocol. The plasmid of interest was incubated in serum free media,and Lipofectaminewas incubated in serum free media forseparately5minutes. The plasmid and the Lipofectamine mixtures(Table 8)were mixedgentlyand incubated at room temperature for 20 min.;thetransfection mixture was added dropwise to the cells. Transfection media was replaced with the fresh complete medium after 6 hrs.of transfection and cell are harvested after 24 hours
    6. Plasmid transfection using Lipofectamine 2000
    7. 293T cells or HeLa cells were transfected with various plasmids as per the designed experiments using Lipofectamine 2000 (Invitrogen) reagent or PEI
    8. PlasmidTransfectionof mammalian cells
    1. To study the virulence of Xanthomonas oryzaepv. oryzicolastrains on rice plant two different inoculation methods, syringe infiltration and wound inoculation methods, were implimented. For infiltration method, bacterial suspension comprising of 1 × 108 cells/ml were infiltrated with needleless syringe into leaves of 4 to 6 week-old rice cultivar of susceptible Taichung Native-1 (TN-1) (Hopkins et al., 1992; Wang et al., 2007). Wound inoculation method was carried out by dropping an aliquot of 20 μl bacterial suspension comprised of 1 ×108cells/ml onto fully expanded leaf of 6-8 week green-house grown Taichung Native-1 cultivar of rice, and pricking with sterile needle for facilitating the entry of Xocinside the leaves throgh wound. For inititation of disease symptom, the inoculated plants were incubated in greenhouse with minimum and maximum temperatures of approximately 25 to 30 °C, respectively, and a relative humidity of approximately 60%. Water soaking symptom and lesion development was measured 4 to 10 days after inoculation. Likewise, for infiltration by wound inoculation method, lesion length was measured 14 days after inoculation. In both the cases, no lesions were observed in control experiments in which the leaves were inoculated with sterile wate
    2. Virulence assay on rice plant
    3. BXOR1, ΔrpfFand ΔrpfF(pSC9) strains were grown to OD600of 1 in rich media (PS), PS + 50 μM 2,2’-dipyridyl (DP) and PS + DP + 30 μM FeSO4. RNA was isolated by Trizol (Invitrogen) method as described above. Optimal primer and cDNA concentrations were standardized, and qRT-PCR was performed using ABI 7500 Fast Real-Time PCR system (Applied Biosystems). In brief, 1 μl cDNA, 0.25 picomoles of gene specific primers and 10 μl 2X SYBR GREEN qPCR Mastermix (Qiagen)were mixed in the wells of 96-well PCR plate (Axygen). Final reaction volume was adjusted to 20 μl with nuclease-free water. Transcript levels were quantified with an end-point value known as Ct(cycle thresold) value. Expression of 16S rRNA was used as an internal control. The Ct values defines the number of PCR cycles required for the fluorescent signal of SYBR green dye to cross beyond the background level. Fold-change in transcript expression was determined using following formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCt treated-ΔCt untreatedΔCttreated = Ctvalue for the gene of interest under treated condition -Ct value for the internal control gene (16S rRNA) under treated conditionΔ Ctuntreated = Ct value for thegene of interest under untreated condition -Ct value for the internal control (16S rRNA) gene under untreated condition
    4. Primers for real-time PCR analysis were designed using Primer3 plus software and are listed in Table 2.2.For RNA isolation, X. oryzaepv.oryzaewild-type, rpfFmutant, rpfF/CG8 complemented strains were grown in PS medium at 28°C for 28 h at 200 rpm. Similarly, for RNA isolation from X. oryzaepv. oryzicola, the Wild-type
    5. Quantitative real-time PCR
    6. E.coliDH5α strain was transformed with plasmids carrying appropriate inserts to generate clones, and Xanthomonas deletion strains. Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 5 μl ligated plasmid was added to 100 μl ultracompetent cells and incubated on ice for 30 min. Next, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred on ice for 2-3 min. Next, 1 ml LB medium was added and cells were allowed to recover for 1 h on a shaker incubator set at 37°C. After the recovery, cells were centrifuged at 3000 g for 3 min. Medium supernatant was discarded and cells were resuspended in 100 μl fresh sterile medium. Cells were plated on LB agar containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 h
    7. E.colitransformation
    8. Liquid scintillation cocktail5 g PPO (2,5-diphenyloxazol)0.3 g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene Volume was adjusted to 1L with toluene.MUG (4-methylumbelliferyl β-d-glucuronide)extraction buffer1 mM MUG substrate50 mM Sodium dihydrogen phosphate (pH-7.0)10 mM EDTA0.1% Triton X-1000.1% Sodium lauryl sarcosine10 mM β-MercaptoethanolLactophenol solution (100 g)25 g Lactic acid (20.66 ml)25 g Phenol 50 g Glycerol (39.77 ml)These three components were mixed together and 1 volume of lactophenol was added to 2 volumes of ethanol
    9. CAS solutiona) 0.06 g Chrome Azurol S dye in 50 mlb) Fe (III) solution: 10 ml1 mM FeCl310 mM HClc) 0.072 g HDTMA in 40 mlAll the above three solutions were mixed together and autoclaved prior to use
    10. Other solutions
    11. Transformation buffer I (Tfb-I) 30 mM Potassium acetate100 mM Rubidium chloride (RbCl2)10 mM Calcium chloride dihydrate (CaCl2.2H2O)50 mM Manganese chloride tetrahydrate (MnCl2.4H2O)15% (v/v) GlycerolpH was adjusted to 5.8 with 10% acetic acid and volume was adjusted to 500 ml with H2O. Transformation buffer II (Tfb-II) 10 mM MOPS75 mM CaCl2.2H2O10 mM RbCl2.2H2O15% GlycerolpH was adjusted to 6.5 with KOH (Potassium hydroxide) and volume was adjusted to 100 ml with H2O.
    12. Buffers for E. colielectrocompetent cell preparation
    13. 50 mM Phosphate citrate buffer (pH-6.8)0.1M Citric acid0.2M dibasic Sodium phosphate16.9 ml Citric acid (0.1 M) and 33.1 ml Sodium phosphate (0.2 M) was mixed and volume was adjusted to 100 ml with H2O.Lipase assay0.1M Tris-HCl buffer (pH-8.2)pH was adjusted to 8.2 with HCl. 0.5 mM p-Nitrophenol standard solution8.69 mg p-Nitrophenol was dissolved in Tris-HCl buffer (0.1M) and volume was adjusted to 25 ml to make a final concentration of 25 mM.1volume of the above solution (25 mM) was diluted with 49 volume of 0.1 M Tris-HCl buffer to get a final concentration of 0.5 mM p-Nitrophenol standard solution.p-Nitrophenyl butyrate solution (420 μM)7.3 μl p-Nitrophenol butyrate (F.W. 209.2) 11 mg SDS650 μL Triton-X-100Volume was adjusted to 100 ml with H2O. Mixture was heated to 65°C in a water bath for 15 min, mixed well, and cooled down to room temperature prior to use. It can be stored upto 3 days at 4°C.Xylanase assay5 mg/ml RBB-xylan0.05 M di-Sodium hydrogen phosphate (Na2HPO4)
  3. May 2019
    1. For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity
    2. For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm
    3. Quantitative assay
    1. 5 mg/ml RBB-Xylan was dissolved in 0.05 M Na2HPO4pH-7.5
    2. Buffers for enzyme assaysCellulase assay
    3. 10% APS -30 μlTEMED -3 μlSDS loading buffer (2X)100 mM Tris-HCl (pH-6.8)20% (v/v) Glycerol4% (W/V) SDS0.02% Bromophenol Blue10% β-MercaptoethanolSDS-loading buffer was prepared as 2X stock solution in H2O and used at 1X concentration.SDS-PAGE running buffer14.4 g Glycine3.03 g Tris methylamine1 g SDSDissolved in H2O and volume was adjusted to 1L with H2O.Buffers for western blot analysisTransfer buffer (1 litre)14.4 g Glycine3.03 g Tris methylamine800 ml H2O 200 ml methanolBlocking and wash buffers (PBS-T)5% Fat-free milk0.05% Tween-20Volume was adjusted to 100 ml with1XPBS
    4. Whole cell lysis buffer50 mM Sodium acetate 410 mg Sodium acetate anhydrous was dissolved in 80 ml H2O. pH was adjusted to 5.4 with glacial acetic acid and finally volume was adjusted to 100 ml with H2O.1 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol.Dialysis buffer50 mM Trizma basepH was adjusted to 7.5 by using concentrated HCl.Silver stainingFixing solution50% ethanol10% glacial acetic acid0.05% formaldehydeFinal volume was adjusted with sterile H2O.0.2% Silver nitrate solution (AgNO3)0.2 g AgNO3
    5. 0.075% formaldehyde (37% stock) Dissolved in 100 ml of H2O. Stored at 4°C for 1 hour in brown colored bottle.Developing solution 6% Sodium carbonate (Na2CO3)0.05% Formaldehyde (37% stock)0.02% Sodium thiosulphateStorage buffer50% EthanolSDS-PAGE30% Acrylamide solution29 g Acrylamide1 g Bis-acrylamideAcrylamide solution was prepared in H2O.Resolving gel mix (12%) (10 ml)H2O -3.3 ml30% Acrylamide:Bisacrylamide mix (29:1) -4 ml1.5 M Tris-HCl (pH-8.8) -2.5 ml10% SDS -100 μl10% Ammonium persulphate (APS) -100 μlN, N, N’,N’,-Tetramethylethylenediamine (TEMED) -4 μlStacking gel mix (5%, 3 ml)H2O -2.1 ml30% acrylamide:bisacrylamide mix (29:1) -500 μl1.5 M Tris-HCl (pH-6.8) -380 μl 10% SDS -30 μl