Genomic DNA extraction

T-Cell Receptor Mutation Assay

RS-1 Assay

Flow Cytometric analysis of variant erythrocytes

Monoclonal Antibodies against the Human Glycophorin-

MN Blood group typing

StatisticalAnalysis

Nucleartranslocationofp-Catenin

FlowCytometricanalysis

AnalysisofAP-landNFKBactivatio

Estimationofprostaglandins,PGEzandPGD

Reagents

Reverse Zymograph

Zymography

Reagents

Western Blotting

SOS-PAGE

MTT Assay

AssayofNAD

AktPhosphorylation

ImmunoprecipitationofVEGF

RT-PCR analysis

Separation ofCurcuminoids

Modified Enzyme Linked Immunosorbent Assay

Enzyme Linked ImmunosorbentAssay

Chorioallantoic membrane (CAM) assay

Aortic ring assay

Isolation of endothelial cells from rat aorta

Isolation and culture of HUVECs

Reducing powe

Procedur

Reagents

Nitric oxide radical scavenging activity

Procedur

Reagents

Hydroxyl radical scavenging assay

Procedure

Reagent

Superoxide anion scavenging activit

Procedure

Reagent

Total antioxidant activit

Procedur

Reagen

Free radical scavenging activity

In VitroStudies

Single Cell Tes

Electrochemical Measurement

Catalyst Preparation

Materials

Single Cell Tes

Electrochemical Measurement

Physical Characterizatio

Catalyst Preparation

Material

Total alkalinity

Dissolved oxygen and Biological oxygen demand

Total dissolved solids

Multi parameter analyzer

Characterization of embelin isolated from E.ribes

. Extraction and isolation of embelin from E. ribes

High Performance Liquid Chromatography (HPLC) analysis

High Performance Thin Layer Chromatography (HPTLC) analysis

. Thin Layer Chromatography (TLC) analysis

Preliminary phytochemical screening (Ali, 1998; Evans, 2002)

Microbial Contamina

Foaming Index

pH values

Ash values

Extractive value

. Physico-chemical standardization

Standardization of ethanolic extract of E.ribes (WHO, 1998; IndianPharmacopoeia, 1996)

Preparation of ethanolic extract of E.ribes

PLANT MATERIAL

Estimation of Stevioside

Extraction from the plant material

Extraction from the plant material

Extraction from the plant material

Qmmtification of stevioside

Estimation of Steviol

Extraction from plant material

Quantification of steviol

Estimation of free aminoacids

Estimation of soluble proteins

Estimation of sugars

Estimation of total phenols

Determination of moisture

Analytical methods

The experimental part of the study was categorized into five sections for the convenience of reference viz. analytical, toxicological, molecular, biochemical and genomic quantitation.

Per-cent Overlap

Compogram

F-and t-Ratios

Chi-Square

Somatotype Categories

Comparative Statistics

Somatotype Attitudinal Mean (SAM)

Somatotype Attitudinal Distance (SAD)

Somatotype Dispersion Mean (SDM)

Somatotype Dispersion Distances (SDD)

Somatoplot Coordinates

Mean Somatotypes (5)

Descriptive Statistics

Statistical Analysis

Central:

Mesomorphic ectomorph:

Endomorphic ectomorph:

Ectomorphic mesomo~ph:

Endomorphic mesomorph:

Ectomorphic endomorph:

Mesomorphic endomor~:

Endomorph-ectom9££E:

Mesomorph-endomorph:

Mesomorph-endomorph:

Balanced ectomorph:

Balanced mesomorph:

Balanced endomorph:

SOHATOCHART

Somatotyping Children

Limitations of the Rating Form

Third Component Rating

Second Component Rating

First Component Rating

SOMATOTYPING

Calf

Biceps

Femur

Humerus

Calf

The DFD between an atom pair is the normalized frequency distribution of the interatomic distances sampled from equal time snapshots taken from the MD simulations. DFDs of corresponding atoms taken from the different simulations were used to qualitatively compare the effect of mutational perturbations on the HbS fiber. Considering that the fiber simulations were carried out only for a relatively short time scale of 1.2ns, the calculated DFDs might suffer from errors due to limited sampling. Hence a quantitative comparison of the different DFDs were not attempted, however given that the global parameters monitored during the simulation had already become reasonably stable after 0.2ns (Chapter!, Table 5), it is expected that the gross features of the DFDs would remain unaltered even in much longer simulations.

Distance Frequency Distribution (DFD)

The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.

luctuation Maps

chains were generated in the same way as for the isolated a-chains. Thus the model consisted of eight polypeptide chains ( 4 a-chains and 4 13-chains) with the central two a and two 13 chains making up the axial contact interface. The simulations of the above fiber models were carried out without explicit solvent using the GROMOS96 vacuum force field as implemented within the GROMACS suite of programs. Models of HbS mutants were generated using the program SCWRL and the mutant fiber models were generated as for the native HbS model. SCWRL replaces only the side chains of desired residues with the best possible rotamer of the mutated amino acids. Thus the initial backbone conformation of the isolated a-chains and the fiber models remained identical in the native HbS and the mutants. The MD simulation protocol for the fiber models consisted of an initial steepest descent energy minimization for 1000 steps followed by full MD simulation for 1.2ns at 300 K. Essential parameters of the simulation like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies are summarized in Chapter I, Table 4. It was observed that all the global indicators of the simulation stabilized to their average values within 0.2ns. Hence data from 0.2ns till the end of the simulations were used for all subsequent analysis

MD simulations on the a-chain of HbS and the mutants were carried out using the GROMACS suite of programs (Lindahl eta!, 2001). The initial structure of the native protein was taken from the high-resolution x-ray crystal· structure (Harrington et a!, 1997) of HbS (PDB entry: 2HBS). The structures of the mutants were generated interactively using INSIGHT-II. The initial model structures were placed in a simulation box of size 42.8 x 31.5 x 42.7 A. The closest distance from any protein atom to the walls of the box was not less than 9 A. The system was then solvated by adding a bath of SPC (Berendson eta!, 1981) waters in such a way that the density of the system was as close to 1 as possible. The overall charge of the system was neutralized by placing suitable counter ions wherever necessary (Chapter 1, Table 4). The resulting system was then energy minimized for 1000 steps using the steepest descent algorithm. This was followed by 0.3ns of position restrained MD during which the solvent and counter ions were allowed to move freely but the protein atoms were harmonically restrained to their initial positions. Finally, normal MD was run for 3ns using the default GROMACS force field. Bond lengths were restrained to their equilibrium values using the LINCS (Hess et a!, 1997) algorithm and a cut-off radius of 0.9nm was used for non-bonded interaction calculation. The temperature of the system was maintained close to 300K by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The integration time step used throughout the simulation was 1 fs. MD simulations of single mutant a-chains (K 16Q, E23Q and H20Q) as well as the double mutants (K16Q/H20Q, K16Q/E23Q and H20Q/E23Q) were carried out in a similar fashion for 3ns. In order to directly analyze the effects of the mutations on the contact interface, we also carried out MD simulations on a miniature model of the sickle hemoglobin fiber consisting of a complex of two hemoglobin tetramers. In the low salt crystal structure of deoxyhemoglobin S (Harrington et a!, 1997) the asymmetric unit consists of two HbS tetramers that pack as two strands of HbS molecules running parallel to the crystallographic a axis. Axial contacts occur between two tetramers of the same strand and lateral contacts occur between tetramers of different strands. A canonical fiber model was generated by taking one of the two tetramers in the asymmetric unit together with its neighbor translated along the crystallographic a axis [Figure 7 (A, B)]. Fiber models incorporating mutant a

Molecular dynamics (MD) simulatio

Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1

Electrostatic Potentia

The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces

Kinetics of polymerization

with the plunger of a Hamilton syringe. The tube was centrifuged at room temperature at 14,000 rpm for 30 min. The above process of gel-disruption and centrifugation was repeated twice subsequent to which the oil layer was aspirated and suitable aliquots from the supernatant were taken for estimation of Csat by Drabkin' s reagent (Goldberg eta!, 1977).

he gelation concentrations of HbS constructs were determined by the dextran-Csat method of Bookchin et a! (Bookchin et a!, 1999). This method allows measurement of Csat under near-physiological conditions and at much lower concentration of HbS (about 5-fold or less) than that required in standard Csat assays, but essentially provides the same information. Briefly, a suitable aliquot of a concentrated solution of hemoglobin in potassium phosphate buffer (0.05 M, pH 7.50) was taken in a 1.5 ml micro-centrifuge tube. A concentrated dextran (70 kDa) solution prepared in the same buffer was added to it and mixed well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on ice bath and deoxygenated with an anaerobically prepared dithionite solution through an airtight Hamilton syringe. The final concentrations of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M respectively. The deoxygenated sample above was allowed to polymerize at 37°C for 30 min after which the gel under the oil layer was disrupted

Measurement of gelation concentration, Csat

All experiments were carried out on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics, and an An60-Ti rotor, at 20 °C. Sedimentation velocity experiments were performed at 40,000 rpm. Data were collected at 540 nm and at a spacing of 0.005 em with three averages in a continuous scan mode. The protein concentration varied in the range 4-40 IJ.M (heme) in 50 mM phosphate buffer, pH 7.2

Analytical Ultracentrifugation experiments

acid. The respective buffer baselines were subtracted from the sample CD data. The ellipticity of the protein samples is reported as mean residue ellipticity (MRE) in deg/cm2/dmol units. The first derivative UV spectra of the oxy and deoxy-HbS were recorded on a Lambda Bio20 spectrophotometer (Perkin Elmer Life ScieAces). The hemoglobin concentration used for the spectral measurements was approximately 50 )!M on heme basis.The spectra of unliganded proteins was recorded subsequent to deoxygenating the hemoglobin samples by passing moist gaseous nitrogen extensively over the sample in an airtight cuvette. Completion of deoxygenation was ascertained by recording the visible spectrum of the deoxygenated Hb sample.

Circular dichroism (CD) spectra were recorded on a J71 0 Spectropolarimeter (Jasco, Japan) fitted with a Peltier type constant temperature cell holder (PTC-348W). The calibration of the equipment was done with (+)-! 0-camphorsulfonic

Spectroscopic studies

The synthetic peptides were purified by RPHPLC on an Aquapore RP300 column (250 x 7 mm) using a 4-72% linear gradient of solvent B (acetonitrile containing 0.1% TFA) in 130 min at a flow rate of 2 mllmin. Globin chains from respective hemoglobins were separated on a similar column of a smaller dimension (250mm x 4.6 mm) under identical conditions but at a flow rate of0.7 ml/min. Electro spray mass spectrometric analysis was carried out on a VG Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard horse heart myoglobin or gramicidin S solution. Appropriate amount of each sample was taken in 50% acetonitrile containing I% formic acid and analyzed under the positive ion mode.

nalytical procedures

Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.

Preparation of HbS{des arg 141a

concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column

Construction of mutant a globins

Reconstitution of a globin and rf chain into HbS tetramers

The semisynthetic a globin was purified from the mixture by CM52 urea chromatography as explained below. The lyophilized sample was dissolved in 0.005 M phosphate buffer (pH 6.9) containing 8 M urea and 0.05 M 2-mercaptoethanol at a concentration of I 0-15 mg/ml and loaded onto a CM52 column (16 em x 1.5 em) equilibrated with the same buffer. After an initial wash with the same buffer, two linear gradients of(a) 100 ml each of0.005 M to 0.03 M and (b) 100 ml each of0.02 M to 0.05 M phosphate buffer (all buffers contained 8 M urea and 0.05 M 2-mercaptoethanol, and were adjusted to pH 6.9) were employed at a flow rate of 45 ml/h to elute the semisynthetic a globin. The column was finally washed with 0.05 M buffer to elute unreacted a31-141 fragment from the column. The elution profile was monitored at 280 nm. The fractions for semi-synthetic a globin were pooled, extensively dialyzed against 0.1% TF A and lyophilized. The semisynthetic yield of the protein varied between 35% to 45%

Purification oftlte semi-!)yntltetic a globin

V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized

Construction of mutant a globins

Peptides were synthesized by standard solid phase synthesis protocols using Fmoc chemistry on a semi-automated peptide synthesizer (Model 90, Advanced Chemtech). For this, Wang resin pre-loaded with N-a-Fmoc-Glu was used as the starting material. The stepwise coupling of Fmoc amino acids was performed with DIPCDIIHOBT activation procedure. The coupling of each step was monitored by Kaiser test for free amine and wherever necessary, a double coupling was used to increase the yield. Before each coupling step and on completion of the synthesis, the N-terminal Fmoc group was removed using 20% piperidine (v/v in DMF). The peptides were cleaved from the resin and the side chains deprotected with appropriate volume of a mixture containing TF A, ethanedithiol, phenol, thioanisole and water (80:5:5:5:5, v/v). The resin was removed by filtration and the crude cleaved peptides were precipitated using cold diethyl ether and extracted in water. The peptides were purified by RPHPLC and their chemical identity was checked by mass spectrometry

ynthesis of al-30 analogs

The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized

Generation of complementary fragments, al-30 and a31-141, from heme-free a globin

The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded

Preparation of heme-free a chain

The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use

The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.

Separation of the a and f3 subunits of hemoglobin

Blood was drawn from appropriate source into heparinised tubes. The blood sample was centrifuged at 4000 rpm for 15 min ( 4 °C). The supernatant was discarded, and the erythrocytes (pellet) were subsequently washed thrice with chilled isotonic buffer [0.01 M PBS (pH 7.4)] by centrifugation at 4000 rpm and 4°C for 15 min. The washed erythrocytes were lysed in water. The resultant red cell lysate was then dialyzed extensively against PBS (pH 7.4) at 4°C to obtain stripped hemoglobin (hemoglobin devoid of bound allosteric modulators like BPG). The stripped hemoglobin was then loaded onto a pre-equilibrated DE52 column (30cm x 15cm) after extensive dialysis against 0.05 M tris acetate buffer (pH 8.5). The protein was eluted from the column employing a linear gradient of 500 ml each of 0.05 M tris acetate (pH 8.5) and 0.05 M tris acetate (pH 7) at a flow rate of 50 ml/hour. The purified hemoglobin was estimated spectrophotometrically at 540 nm (molar extinction coefficient= 53236 cm-1/M) and stored at -70°C till further use.

Purification of hemoglobin from bloo

Methods

Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads

Flow Cytometry

The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field

Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.

For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m

Soft Agar Assay

For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning

Clonogenic Assay

and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.

4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS

Procedure:

ml 1X PBS

1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF

Working Solutions:

20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7

Stock Solutions:

This protocol is for the detection of β-gal expression in fixed cells. It was performed on 96-well plates for initial screening of tTA transfected clone, and is a modification of Sanes et al., 1986

In situβ-gal staining of Transfected Cells

Procedure:

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

Solutions:

β-gal assay in transfected cells

normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice

Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were

Growth Curves:

After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA

The PCR parameters were as follows

The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).

The following K-Ras-ras mutants were generated by site directed mutagenesis according to the protocol described in QuickChange site directed mutagenesis kit (Stratagene). The primers used are shown in the following table