Vaccination schedule and assessment of parasitic burden

Solutions used for cytokine assay

Assessment of Cytokine levels- IFN--12/IL-10 in lymphocytes of cured/endemic patients

Preparation of soluble L. donovani promastigote antigen

Polyclonal antibody generation

Recombinant protein Expression

Sequencing of ADHT gene

Restriction Digestion of Plasmid DNA

Colony PCR

Preparation of master plate and isolation of plasmid DNA from transformed E. coli (Mini Prep)

Confirmation of positive clone(s)

Elution of amplified gene from the Gel

Evaluation of QSAR models

Inhibitors Dataset

Molecular Docking

Assay for reduced glutathione(GSH)

Acridine orange/ethidium bromide (AO –EB) staining

Superoxide radical scavenging activity

FTIR analysis

GC-MS analysis

Algorithm

EM Procedure

Empirical Bayesian Smoothing

Haemocyte morphogenesis

Spreading inhibitory behavior

Inhibition of haemocyte aggregation

Haemolymph protein profile

Total haemocyte count assay

Immunomodulatory

Helicoverpa armiger

Spodoptera litura

Gut enzyme profile

Insecticidal activity

. Statistical analysis

Inhibition of haemocytes spreading behavior

. Inhibition of haemocytes aggregation behavior

Haemolymph protein profiling

Total haemocyte count (THC)

Haemolymph collection

Immunomodulatory

Lactate dehydrogenase

Asparate (AAT) and Alanine aminotransferase (ALT)

Gut enzyme profile

Oral toxicity bioassay

Microinjection bioassay

VS preparation

Insect collection

INSECTICIDAL AND IMMUNOMODULATORY ACTIVITY AGAINST INSECT PEST

Quantification of protein of VS yielded in the prey deprivation

Venomous saliva utilization

Maxillary stylet

Morphometry of head and stylet

Lactic dehydrogenase

Cholesterol content

Characteristic features ofLabeo rohita

Animal model for the study-Labeo rohita

Residual nutrients

Organic carbon

Soil sampling

Biomass and harvest inde

Yield and its component traits:

Phenological traits and plant height

Regression analysis

Statistical analysis

Kernel hardness

Coleoptile length(cm)

Plant height (cm)

pH

Identification of the strains

Tested individually

Increased Chemokine and Toll like receptor on T cells after vaccination in HBV positive newborns.

Cord Blood immune profiles of HBV positive newborns at birth(Cord blood vs. peripheral blood)

CD107a expression (marker of cytotoxicity)

HBV vaccination of the newborn

1X TBST

Immunoprecipitation and western Blotting

Medium and Serum

GFP-β-catenin- a.a

Cell Viability and IC50determination byMTT assay

Bacterial Strain

Cell Lines

Bacterialstrain

Procedure

Estimation

Calculation

Extraction and determination of sugar

Standard curve of sugar

Reagents

Estimation of total sugar

Extraction and determination of protein

Extraction and determination of ascorbic acid

LC 50 value

Dilution Formula

FACS buffer

Statistics

Cell lines

Plasmid mini preparation by alkaline lysis

X-gal

Flow Cytometric analysis of variant erythrocytes

Flow Cytometric analysis of variant erythrocytes

Aortic ring assay

Procedure

Reagent

Superoxide anion scavenging activit

Procedure

Physical Characterizatio

Single Cell Test

Electrochemical Measuremen

Physical Characterization

Catalyst Preparation

Materials

ASSESSMENT OF SEAWATER ELECTROLYSIS ON PLATNIZED TIN AND MAGNESIUM ALLOY NANOPARTICLES FORNON-MEMBRANE POWER SYSTEMS

Physical Characterization

Dissolved oxygen and Biological oxygen demand

. Extraction and isolation of embelin from E. ribes

High Performance Thin Layer Chromatography (HPTLC) analysis

pH values

Estimation of soluble proteins

Per cent Overlap:

somatotype Categories:

Mean Somatotypes:

Age Changes in' Somatotypes

Range of Component Ratings:

Range of Component Ratings:

Balanced ectomorph:

Isolation of inclusion bodies from E. coli cells

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

enginethatistheproblembut,rather,theusersofsearchengineswhoare.Itsuggeststhatwhatismostpopularissimplywhatrisestothetopofthesearchpile

'Sam!' he called. 'Pippin! Merry! Come along! Why don't you keep up?'10There was no answer. Fear took him, and he ran back. As he struggled on he called again, and kept on calling more and more frantically. He was weary, sweating and yet chilled. It was wholly dark.'Where are you?' he cried out miserably.There was no reply. He stood listening. He was suddenly aware that it was getting very cold, and that up here a wind was beginning to blow, an icy wind. A change was coming in the weather. The mist was flowing past him in shreds and tatters. His breath was smok­ing.11 He looked up and saw with surprise that faint stars were ap­pearing overhead amid the strands of hurrying cloud and fog. Oat of the east the biting wind was blowing.'Where are you?' he cried again, both angry and afraid.'Here!' said a voice, deep and cold, that seemed to come out of the ground. 'I am waiting for you!''No!' said Frodo; but he did not run away. His knees gave,12 and he fell on the ground. Nothing happened, and there was no sound. Trembling he looked up in time to see a tall dark figure like a shadow against the stars. It leaned over him. He thought there were two eyes, very cold though lit with a pale light that seemed to come from some remote distance. Then a grip stronger and colder than iron seized him. The icy touch froze his bones, and he remembered no more.When he came to himself again, for a moment he could recall nothing except a sense of dread. Then suddenly he knew that he was imprisoned, caught hopelessly; he was in a barrow. A Barrow-wight had taken him, and he was probably already under the dreadful spells of the Barrow-wights about which whispered tales spoke. Hedared not move, but lay as he found himself: flat on his back upon a cold stone with his hands on his breast.As he lay there, thinking and getting a hold on himself, he no­ticed all at once that the darkness was slowly giving way:13 a pale greenish light was growing round him. He turned, and there in the cold glow he saw lying beside him Sam, Pippin, and Merry.There was a loud rumbling sound, as of stones rolling and fal­ling, and suddenly light streamed in. A low door-like opening appeared at the end of the chamber beyond Frodo's feet; and there was Tom's head against the light of the sun rising red behind him.'Come, friend Frodo!' said Tom. 'Let us get out on to the clean grass! You must help me bear them.' Together they carried out Merry, Pippin and Sam. To Frodo's great joy the hobbits stirred, robbed their eyes, and then suddenly sprang up. They looked about in amazement. 'What in the name of wonder?14 began Merry. 'Where did you get to, Frodo?''I thought that I was lost', said Frodo; 'but I don't want to speak of it.' But Tom shook his head, saying: 'Be glad, my merry friends, and let the warm sunlight heat now heart and limb! Cast off these cold rags! Run naked on the grass!'

A stock solution of xylose (1 mg mL-1) was prepared in distilled water. A dilution series ranging from 100-1000 μg mL-1 was prepared from the stock solution. To 1 mL of solution, 1mL of DNSA was added and kept in a boiling water bath for 10 min and then 400 μL of sodium potassium tartrate solution was added and kept it for cooling. The absorbance was recorded in a spectrophotometer (Shimadzu, UV-VIS) at 540 nm

Preparation of standard curve of xylose

Transformation of calcium-competent cells was carried out by the procedure detailed below: •The competent bacterial cells were thawed briefly and 200 μL of cells was mixed rapidly with plasmid DNA (10-50 ng) in fresh, sterile microcentrifuge tubes and maintained on ice for 30 min. A negative control with competent cells only (no added DNA) was also included. •Cell membranes were disrupted by subjecting cells to heat-pulse (42 °C) for 90 sec. •After heat shock, cells were incubated on ice for 5 min. •Cells were then mixed with 1 mL LB medium and incubated with shaking at 37 °C for 1 h. •For blue/white screening 40 μL of X-gal solution (20 mg mL-1 in dimethylformamide) and 4 μL of the IPTG (200 mg mL-1) was spread on LB-ampicillin (LB-amp) plates with a sterile glass rod. The plate was allowed to dry for 1h at 37 °C prior to spreading of bacterial cells. •Bacterial cells (100-200 μL) were spread and the plate was incubated at 37 °C for overnight. •White colonies were picked from the plates and suspended into LB-amp broth and cultivated to OD600=0.5

Transformation procedure

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

Ligation of insert DNA with dephosphorylated vector

In order to minimize self ligation of vector during cloning experiments, the digested DNA was subsequently treated with calf intestinal phosphatase (CIP) [NEB, UK]. The reaction conditions and amount of CIP were optimized and varied from (0.06-1) unit/picomole DNA termini. The dephosphorylation reaction was carried out in 50 μL reaction as follows. Reaction mixture containing no restriction enzyme was treated as control. Reaction was incubated for 1 h at 37 °C and stopped by heat inactivation at 65 °C for 20 min. 2.5.5. Composition of restriction mixture (50 μL) Linearized Plasmid DNA X μL (1 μg) CIP 1 μL (0.06-1 U μL-1) Reaction buffer (10X) 5.0 μL Distilled water Y μL Total volume 50 μL Linearized and dephosphorylated plasmids from each reaction were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany). 100 ng DNA from each reaction was then ligated in15 μL reaction volume containing 1.5 μL of 10X ligation buffer (NEB, England) and 0.2 μL of T4 DNA ligase to check the efficiency of self ligation after dephosphoryaltion. The ligation mixture was incubated at 16 °C for overnight and transformed into E. coli DH5αcompetent cells.

Dephosphorylation of the restricted plasmid

The vector isolated as above was digested with BamHI to generate the cohesive ends. The reaction was performed in 1.5 mL Eppendorf tubes as described below. Composition of restriction mixture (100 μL) Plasmid DNA X μL (20 μg) Bam HI 8 μL (10 U μL-1) NEB buffer 4 10.0 μL BSA (100X) 1 μL MQ water Y μL The reaction mixture was incubated at 37 °C for 3 h. The digestion was stopped by heat inactivation at 65 °C for 20 min. The digestion of plasmid was checked using 1.2 % (w/v) agarose gel electrophoresis for linearization of the plasmid. The digested plasmid was purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany).

Restriction digestion of plasmid DNA

Two hundred μL of alkaline-SDS solution was added to the above suspension, mixed by inverting the tubes up and down 3 times and incubated for 5 min at room temperature. ƒTo the above mixture, 250 μL of 3 M Na-acetate (pH 4.8) was added, mixed by inverting the tubes up and down 3 times, and centrifuged at 12,000 x g for 10 min. ƒThe supernatant was collected in another micro centrifuge tube (MCT), 200 μL of phenol:chloroform solution was added, inverted two times and centrifuged at 12, 000 x g for 8 min at room temperature. ƒThe aqueous phase was transferred to new tubes and 500 μL of chilled (-20 °C) ethanol (96 %) was added. ƒThe tubes were centrifuged at 13,000 x g for 25 min at 4 °C, supernatant discarded and pellet dried for 15 min at room temperature. ƒThe pellet was washed with 500 μL of chilled 70 % (v/v) ethanol and centrifuged at 13, 000 rpm for 4 min at 4 °C. ƒThe pellet was dried at room temperature and dissolved in 50 μL of 1X TE buffer (pH 8.0) containing RNase and stored at -20 °C till further use.

The cells of E. coli DH10B having p18GFP vector were cultivated for overnight at 37 °C in LB medium containing ampicillin (100 μg mL-1). ƒThe E. coli culture having p18 GFP vector (~1.5 mL) was taken in Eppendorf tubes and centrifuged at 10, 000 x g for 5 min. ƒThe pellet was homogenized by vortex mixing in 100 μL of homogenizing solution

Plasmid isolation from miniprep method

The metagenomic DNA extracted from above defined protocol was digested with Sau3A1 at conditions optimized to generate maximum fragment in the size range of 2-6 kb. Different concentration (0.05 to 1 unit) of enzyme was used to optimize the digestion of 1 μg of DNA. Reactions were carried out in a final volume of 30 μl each in an Eppendorf of 1.5 mL. Reaction mixture (1 μg DNA having 3 μL NEB buffer 3 and 0.3 μL of 10X BSA) were kept at 37 °C for 10 min and stopped by heat inactivation at 80 °C for 20 min. Different digested reactions were checked for the desired fragments using 0.8 % (w/v) agarose gel electrophoresis. After optimization of DNA fragments for the appropriate size, a large scale digestion was carried out and the fragments (2-8 kb) were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany)

Insert DNA preparation

CONSTRUCTION OF METAGENOMIC LIBRARY

An attempt was made to study the effect of storage of DNA extracts on DNA yield and purity. The DNA extracts were centrifuged and the supernatants were dispensed into 2.0 mL Eppendorf tubes and stored at -20 oC for a month. DNA precipitation and its quantification were carried out at a week intervals.

Effect of storage on soil/sediment DNA extracts

The isolated DNA was diluted (1:100) with MQ. The concentration (mg mL-1) of the DNA [N] was determined spectrophotometrically by recording absorbance at 260 nm (A260) as: A260 = ε 260[N]where ε 260 is the extinction coefficient of DNA (50 for ds DNA) [N] = concentration (mg mL-1) of DNA The concentration of ds DNA [N] was calculated as [DNA] (mg mL-1) = A260/ε 260 [DNA] (μg mL-1) = A260 × 50 × dilution factor Purity of DNA was checked by measuring absorbance at 260 and 280 nm and calculating the A260/A280 ratio (Sambrook et al., 1989). A DNA sample was considered pure when A260/A280 ranged between 1.8-1.9. An A260/A280 < 1.7 indicated contamination of the DNA preparation with protein or aromatic substances such as phenol, while an A260/A230 < 2.0 indicated possible contamination of high molecular weight polyphenolic compounds like humic substances.

Determination of DNA quantity and purity

as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.

The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998

Alternatively metagenomic DNA was extracted from the alkaline soil samples by using different commercial kits (UltraClean™, PowerSoil™ [Mo Bio Laboratories Inc., Carlsbad, CA, USA], Nucleospin kit [Macherey-Nagal, Germany] and Zymo soil DNA isolation kit [CA, USA]). The DNA was finally suspended in 100 μL of sterile Milli Q water for further analysis.

Commercial kits

Comparison of yield and purity of crude DNA

Soil (1 gm) was suspended with 0.4 gm (w/w) polyactivated charcoal (Datta and Madamwar, 2006) and 20 μL proteinase K (10 mg mL-1) in 2 mL of modified extraction buffer [N,N,N,N cetyltrimethylammonium bromide (CTAB) 1% w/v, polyvinylpolypyrrolidone (PVPP) 2% w/v, 1.5 M NaCl, 100mM EDTA, 0.1 M TE buffer (pH 8.0), 0.1M sodium phosphate buffer (pH 8.0) and 100 μL RNaseA] [Zhou et al., 1996] in 20 mL centrifuge tubes to homogenize the sample and incubated at 37 °C for 15 min in an incubator shaker at 200 rpm. Subsequently, 200 μL of 10% SDS was added to the homogenate and kept at 60 °C for 2 h with intermittent shaking. DNA was precipitated by adding 0.5 V PEG 8000 (30 % in 1.6 M NaCl) and left at room temperature for an hour (Yeates et al., 1998). The precipitated DNA was collected by centrifugation at 8000 x g at 4 °C. The supernatant was discarded and pellet was dissolved in 1 mL of TE buffer (pH 8.0) and then100 μL of 5 M potassium acetate (pH 4.5) was added and incubated at 4 °C for 15 min. The supernatant was collected after centrifugation at 8000 x g and treated with equal volumes of phenol: chloroform (1:1) followed by chloroform: isoamylalcohol (24:1) at 8000 x g for 15 min

PROTOCOL FOR OPTIMIZATION OF HUMIC ACID-FREE DNA FROM ALKALINE SOILS

cycles at 94°C for 30 s, 45°C for 30 s, 68°C for 2 min and final extension at 72°C for 10 min (see table 3.1). PCR products were cloned in pGEM-T easy vector (Promega) and the sequence for the cloned PfCDPK4 gene was obtained by automated DNA sequencing

The PCR reaction was carried out usmg Hi-fi Platinum Taq polymerase (Invitrogen) and primers PfCDPK4_F and PfCDPK4_R (see list II) with the following cycling parameters: 94°C for 2 min initial denaturation followed by 3

To obtain PfCDPK4 gene sequence, BLAST search was done usmg either i TgCDPK1 or the published sequence of other CDPKs in the P. Jalciparum genome sequence. An ORF on chromosome 7 exhibited significant sequence homology with other PfCDPKs. Subsequently, PlasmoDB annotation appeared in the public domain and the gene sequence PF07 _0072 matched with the PfCDPK4 sequence! identified by us. For PCR amplification, primers were designed on the basis of' nucleotide sequence of PFb7 _0072. Total RNA from asynchronous P. Jalciparum, cultures was isolated using RNA easy Kit (Qiagen, Germany) and was used to' synthesize cDNA for reverse transcription (RT). Both complimentary and genomic DNA were used as template.

Molecular Cloning of PfCDPK4

For cryopreservation of P, Jalciparum cultures, mostly ring stage parasites at a high parasitemia were obtained. The parasites were pelleted by centrifugation at 200 g for 5 min with minimum de-acceleration. To the pellet, 1.5 volume of the freezing solution (list I) was added drop-by-drop, while shaking the vial gently; the ad4ition was completed in ~ 1 min. The medium was then transferred into a sterile cryovial, which was stored in ~he liquid nitrogen tank

Cryopreservation of Plasmodiumfalciparum cultures

sodium sulphate, filtered and concentrated. The product was reconstituted in methanol and desalted using LH-20 sephadex column. The identity of the aldehyde was confirmed by TOF-MS and tandem mass spectrometric analysis using ESI-MS (API QST AR Pulsar i MSIMS, Applied Biosystems). Purification of peptide was performed using RP-HPLC. The aldehyde could be resolved from other impurities (including traces of alcohol, valeryl-FTAAlaninol) on Cl8 RP-HPLC column (7.8 x 300 mm, 125A, Waters) using a gradient of 0-48% B in 20 mins, 48% B in 40 mins and 90%B in 50 mins (A: water with 0.1% TF A and B: acetonitrile with 0.1% TF A) using a flow rate of 2 mllmin. The elution profile was monitored at 220 nm and the identity of the aldehyde was confirmed by mass spectrometric analysis

The aldehyde valery I-L-Phe-L-Thr-L-Ala-L-Alaninal (valery!-FT AAlaninal) was synthesized by Fmoc-solid phase solid phase chemistry using the Weinreb AM resin (Novabiochem, 0.63 mM/g) and automated peptide synthesizer (Advanced Chemtech. USA). Fmoc protecting groups of amino acids were removed by 20% piperidine in double distilled dimethyl formamide (DMF). A fourfold excess of respective amino acids were preactivated using HoBt (2 equivalents) in DMF and the coupling was catalyzed by diisopropylcarbodiimide (DIPCDI, 2 equivalents). After synthesis resin was dried with dichloromethane/DCM (3 X) and MeOH (3 X). The Thr side chain protecting group (tertiary butyl) was removed by treatment with 60:40, TF A: DCM, twice. The filtrates were discarded and resin was washed with DCM (3 X) and MeOH (3 X). The dried resin was suspended in tetrahydrofuran (3 ml) in a glass reaction flask (25 ml) under nitrogen, swelled with gentle stirring for 1 h, and then cooled to 0 °C. Cleavage of the peptide aldehyde from the resin was performed by adding lithium aluminium hydride (Aldrich. 2 M equivalents dissolved in THF) drop wise for 30 min at 0°C with constant stirring. The reaction was quenched with careful addition of KHS04 (saturated solution) and stirred until the solution reached room temperature. The resin was then filtered off and washed with DCM (3 X) and MeOH (3 X). The filtrate was treated with sodium potassium tartrate (saturated solution) and organic layer was extracted. This organic layer was dried over

Clzemical synthesis and purification of aldehyde valeryl-FTA-Aianina

released from the para-ovarian pad of fat as well as from the peritoneal reflections while care was taken to avoid injury to the ovarian vessels. A ligature was tied around the distal end of the fallopian tube including the ovarian vessels following which the ovary was excised. Hemostasis was secured before the stump of the tube was pushed back into the peritoneal cavity. The peritoneum was closed by continuous sutures using 2-0 silk. The same protocol was followed to perform oophorectomy on the contralateral side. The muscular layer and skin were closed together using surgical clips

The anesthetized mice were operated under strict aseptic conditions inside a laminar flow hood. The mouse was placed over layers of sterile tissue paper and the skin overlying the dorsal flanks was sterilized by wiping with 70% ethanol. The flank was palpated gently to identify the kidney, and an incision(~ 5 mm) was made using a pair of scissors on the overlying skin which penetrated the skin, sub-cutaneous tissue and the muscle layer with the parietal peritoneum being exposed and intact. The para-ovarian pad of fat was identified through the intact peritoneum and a small incision was made on the peritoneum overlying it. The ovarian tissue along with the fallopian tube was mobilized and delivered through the incision site. The ovary was

Bilateral oophorectomy

Leishmania major strain (MHOM/Su73/5ASKH) was a kind gift from Dr. Satyajit Rath, Immunobiology Laboratory, National Institute of Immunology, India. L.major promastigotes were cultured at 23°C in modified DMEM (DMEM (1 L) supplemented with sodium bicarbonate (3.7 g), HEPES (5.96 g), hemin (5 mg), biotin (1 mg), adenine (13.36 mg), xanthine (7.6 mg), triethanolamine (0.5 mL), and tween 80 (40 mg)) supplemented with 10% FCS. It is known that long term culture of L.major promastigotes results in loss of their virulence (2). Hence, to maintain the virulence of these parasites, they were propagated in mice footpad. Towards this end, the stationary phase L.major promastigotes were resuspended in Hank's balanced salt solution and 2x106 promastigotes were injected into the footpad of female BALB/c mice. 6 weeks post-infection, the infected footpad was dissected and the lesion harvested. The obtained lesion was minced and resuspended in modified DMEM supplemented with 10% FCS and placed in 23°C incubator to allow differentiation of intracellular amastigotes to promastigotes. This cycle of harvesting promastigotes from footpad lesions was performed every 6 weeks to maintain the virulent phenotype of this parasite

Protocol for propagation and maintenance of Leishmania major promastigotes

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).

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

Automated molecular replacement program (MOLREP)

merging data, and symmetry equivalent positions, space group-specific systematic absences, total percentage of data collected and the linear Rmerge for data reduction. Finally, truncate program was used to obtain structure factor or amplitudes from averaged intensities (output from SCALA, or SCALEPACK) and write a file containing mean amplitudes and the original intensities. If anomalous data is present then F(+), F(-), with the anomalous difference, plus I(+) and 1(-) are also written out. The amplitudes are put on an approximate absolute scale using the scale factor taken from a Wilson plot. For all the Fab-peptide complexes and unliganded Fab of BBE6.12H3 antibody, the diffraction data were collected and processed using MOSFLM and subsequently merged using SCALA. For all the Fab-peptide complexes of 36-65 Fab, the diffraction data were collected and processed using DENZO and subsequently merged using SCALEPACK. The cell dimensions and space groups were unambiguously determined for each crystal. The solvent content and Matthews's constant were calculated (Matthews, 1968). The merged and scaled intensities were used for structure determination.

parameters using the whole data set. It is also used for merging different data sets and carrying out statistical analysis of the measurements related by space group symmetry. SCALEPACK also provides the detailed analysis of the merged data, and symmetry equivalent positions, space group-specific systematic absences, total percentage of data collected and the linear Rmerge for data reduction. MOSFLM is a package of programs with an integrated graphical user interface for processing data collected on any detectors. The programs cover all aspects of data reduction starting from the crystallographic pattern recorded on an image to the final intensities of observed reflections. In MOSFLM this entire process of integration of diffraction images is subdivided into three steps. The first is the determination of the crystal parameters, in particular the crystal lattice (unit cell) and its orientation relative to a laboratory axial system (usually based on the X-ray beam direction and the rotation axis)_ This is usually referred to as autoindexing. Knowledge of these parameters then allows an initial estimate of the crystal mosaicity. The second step is the determination of accurate unit-cell parameters, using a procedure known as post-refinement. This requires the integration of one or more segments of data with a few images in each segment. The final step is the integration of the entire set of diffraction images, while simultaneously refining parameters associated with both the crystal and the detector. After integration of the data, next step is to scale and merge the data set. Scaling and merging are done with the program SCALA. This program scales together multiple observations of reflections, and merges multiple observations into an average intensity. The merging algorithm analyses the data for outliers, and gives detailed analyses. It generates a weighted mean of the observations of the same reflection, after rejecting the out:iers. SCALA also provides the detailed analysis of

therefore only partially recorded on any individual image. For each predicted reflection, the background-subtracted diffracted intensity must be estimated. Although straightforward in principle, defects and limitations in both the sample (the crystal) and the detector can make this difficult in practice. Complicating factors include crystal splitting, anisotropic and/or very weak diffraction, high mosaicity, diffuse scattering, the presence of ice rings or spots, unresolved or overloaded spots, noise arising from cosmic rays or zingers, backstop shadows, detector blemishes, radiation damage and spatial distortion. These experimental factors will be important in determining the final quality of a data set. The HKL2000 (Otwinowski, 1997) is GUI based suite of programs for the analysis of X-ray diffraction data collected from single crystals. The package consists of three programs: DENZO, XDISPLA YF and SCALEPACK. HKL is the program that converts the raw X-ray diffraction data, collected from an image plate and reduces it to a file containing the hkl indices, intensities of the spots on the image plate along with estimates of errors involved. DENZO initially performs peak searching. The autoindexing algorithm carries out complete search of all the possible indices of the reflections picked by peak search using a fast Fourier transformation (FFT) software module. After search for real space vectors is completed, the program finds the three best linearly independent vectors, with a minimal unit cell volume, that would index all of the observed peaks. After refining the initial cell dimensions and detector parameters, the determined values are applied to the rest of the frames and the parameters are refined for each frame. The diffraction maxima are also integrated by DENZO_ The program XDISPLA YF (W., 1993) enables visualization of the peak search and processing procedures. SCALEPACK finds the relative scale factors between frames and carries out precise refinement of crystal

The collection of macromolecular diffraction data has undergone dramatic advances during the last 20 years with the advent of two-dimensional area detectors such as image plates and CCDs, crystal cryocooling and the availability of intense, monochromatic and highly collimated X-ray beams from synchrotron sources. These technical developments have been accompanied by significant advances in the software used to process the resulting diffraction images. In particular, autoindexing procedures have improved the ease of data processing to the point that in many cases it can be carried out automatically without any user intervention. However, the procedure used to collect the diffraction images, the screenless rotation method, has remained essentially unchanged since it was first suggested for macromolecular crystals by Xuong et al. (Nguyen-huu-Xuong, 1968) and by Arndt and coworkers and popularized by the availability of the Arndt-Wonacott oscillation camera (Arndt, 1977; U. W. Arndt, 1973). In this procedure, each diffraction image is collected while rotating the crystal by a small angle (typically between 0.2 and 2°) about a fixed axis (often referred to as the cpaxis). The only development of the method has been the use of very small rotation angles per image (the so-called fine cp-slicing technique) to provide improved signal to noise for weakly diffracting samples. Since, virtually all macromolecular diffraction data are collected in this way (with the exception of data collected using the Laue technique). The starting point for data integration will therefore be a series of such diffraction images and the desired outcome is a data set consisting of the Miller indices (hk/) of all reflections recorded on these images together with an estimate of the diffracted intensities I(hkl) and their standard uncertainties al(hkl). This requires the prediction of which reflections occur on each image and also the precise position of each reflection on each image (note that typically most reflections will be present on several adjacent images and

X-ray intensity data processing

Fab purification from the digestion mixture was carried out by ion-exchange chromatography using SPW-DEAE (60xl50 mm) column on a Waters3000 preparative HPLC (Waters, USA). In:tially, a blank run was carried out thereafter the column was allowed tore-equilibrate with the wash buffer (10 mM Tris-Cl, pH 8.0). A salt gradient of 0 to 0.2 M NaCI over a period of 120 minutes was used to elute the Fab. An aliquot from all the collected fractions were precipitated by using chilled acetone and were analyzed on a SDS-PAGE gel to ascertain which fraction corresponds to Fab. Fab, which has low or zero net negative charge at pH 8.0, was eluted out as the first major peak early in the gradient. The Fe portion and any undigested IgG which have a higher net negative charge at pH 8.0 would elute out later in the gradient. The Fab fractions collected from various HPLC runs for both the antibodies were pooled, concentrated and dialyzed against their respective crystallization buffer (50 mM Na-cacodylate pH 6.7, 0.05% sodium azide and SOmM Tris-Cl pH 7.1, 0.05% sodium azide).

Purification of Fab fragment

band has completely disappeared and only the 25 kDa doublet is clearly visible. Optimal w/w ratios of protein/enzyme and time of incubation were ascertained and preparative digestions were carried out using 20-50 mgs of IgG. The digestion mixture was then dialyzed against 10 mM Tris-Cl, pH 8.0 for Fab purification.

The concentration of the purified IgG was estimated usmg micro bicinchoninic acid (BCA) method of protein estimation (Micro BCA Protein Assay Kit , Pierce). To ascertain the optimal ratio of IgG to papain and time of incubation for proteolytic fragmentation of the antibody, an initial analytical digestion was carried out. 1 mg/ml solution of papain (Sigma, St. Louis, USA) was first pre-activated using rJ-mercaptoethanol (2.0m\1) for one hour. The reaction mixtures for digestion constituted 400 J.lg of IgG, rJ-mercaptoethanol (2.0mM) and EDTA (2.0mM) appropriate volumes of the activated papain solution were added so as to obtain various ratios of lgG:papain. The digestion reaction was monitored for 10 hours and 20 Ill aliquots were collected every hour. The proteolysis reaction was stopped with addition of 75 mM iodoacetamide and all the aliquots were analyzed on reducing SDS-PAGE. Initially the lgG appears as two bands, one at 50 kDa and the other at 25 kDa, corresponding to the heavy and light chains, respectively. The intensity of the 50 kDa band decreases and instead, a doublet starts appearing at 25 kDa as the reaction proceeds. The Fe portion may or may not be visible as a band just above the 25 kDa doublet. The digestion is deemed complete when the 50 kDa

Generation of Fab fragment

solution was injected into the HPLC. A salt gradient of 0 to 0.2 M NaCl over a period of 120 minutes was run and fractions for each peak, as detected by measurement of UV absorbance at 220nm, were collected. An aliquot of each fraction was subjected to acetone precipitation and the obtained precipitate was analyzed on SDS-PAGE to ascertain which fraction corresponds to IgG. The IgG fractions from different runs were pooled and concentrated to -1 mg/ml which was then dialyzed against the digestion buffer (0.15 M NaCl, O.lM Tris-Cl, pH 7.1).

The collected ascitic fluid was centrifuged to remove cell debris and fat. Mouse monoclonal ascites, was filtered through glass wool to remove lipid like material left over after centrifugation. The supernatant was then subjected to (Nr4)zS04 fractionation. Saturated (Nlit)zS04 solution (SAS) at pH 7.0 was gradually added to the ascites in an ice bath with continuous stirring till a concentration of 40% (v/v) was achieved. The mixture thus, obtained was centrifuged to get the protein pellet and the pellet was re-suspended in buffer (0.01 M Tris-Cl, pH 8.5). The crude antibody solution obtained from ammonium sulfate fractionation was dialyzed against the wash buffer (0.0 1 \1 Tris-Cl, pH 8.5) and then subjected to ion-exchange chromatography using 5PW-DEAE (60x150 mm) column on a Waters3000 preparative HPLC (Waters, L:SA), to purify IgG. All solutions used during chromatography were filtered (0.451-lm) and then degassed. Following equilibration of the column with wash buffer, a 2 ml aliquot of the crude antibody

Antibody purification

secrete antibody which gets collected in the peritoneal fluid. Ascites is thus a good source of monoclonal antibody. The hybridoma cells, from two different hybridoma, which were secreting IgGs, 36-65 and BBE6.12H3, were injected into the peritoneal cavity of male Balb/c mice irradiated with a dose of 400 RAD and primed with Freund's incomplete adjuvant 72 hours prior to injecting the hybridoma cells suspended in 500 111 of Dulbecco's phosphate buffered saline (DPBS). Approximately 5 x 105 to 5 x 106 hybridoma cells were injected into each mouse. Ascitic fluid could be tapped from the peritoneal cavity of mice after approximately 4-5 days.

Ascites is the intra-peritoneal fluid collected from mice that have developed peritoneal tumor. Hybridoma cells, when injected into the peritoneal cavity of mice,

Generation of ascites from mice

conditions used for purificatior and crystallization. Therefore, in any attempt at crystallization, the homogeneity of the preparation and its stability in solution is of paramount importance. lgG molecules possess a net negative charge, at pH 8.5. At this pH, IgG molecules, can therefore, bind to positively charged chemical moieties, an association that can be reversed using low concentrations of sodium chloride. Hence, it is possible to purify IgG using anion-exchange chromatography with a salt gradient. Since this method does not involve harsh conditions it represents an ideal way to purify IgG to be ultimately used for crystallization experiments. Ammonium sulphate fractionation prior to chromatography improves the resolution of peaks during IgG purification. The mouse IgG molecules are known to precipitate in the range of 33% to 40% (v/v) saturated ammonium sulphate. lgG molecules have three major domains, each of which are mobile with respect to the other. Consequently, the molecule is highly flexible and not easy to crystallize. Although entire immur.oglobulin molecules have also been crystallized (Harris et a!., 1998), the proteolytic fragments of IgG molecules have been observed to be more amenable to crystallization. Papain, a non-specific proteolytic enzyme obtained from papaya, cleaves antibodies at the hinge region to release one Fe and two Fab domains. Proteolysis by papain, if carried out carefully, followed by purification can yield a homogenous preparation of Fab. This pure preparation of Fab can be used for crystallization experiments.

A prerequisite for collection of X-ray diffraction data for structure determination is availability of well-ordered single crystals. Protein crystallization is considered to be an art and there are no universally applicable methods for obtaining crystals suitable for X-ray diffraction studies. However, it is unlikely that good crystals will not be obtained if attention is paid to the purity and stability of the molecule of interest. A high degree of purity is essential for successful crystallization of most proteins. Also, the molecule must be stable under the

Preparation of Fab fragment

I 0.0 J.Ll 16.0 J.LI I 0.0 J.Ll 4.0J.Ll 4.0J.Ll 0.5 J..ll 55.5 J.LI I 00 J.L

lOX PCR buffer 1.25 J.LM dNTP mix Template DNA (I 0 J.Lg/ml) Primer #I (25 nmoles/ml) Primer #2 (25 nmoles/ml) Taq DNA polymerase (5 U/J.Ll) Sterile water Total volume

The reaction was started by an initial hot start at 94 °C for 5 min., followed by a three-step amplification cycle. The amplification cycle consisted of a 1 min. denaturation at 94 °C, followed by a 2 min. annealing at 48 °C and an extension at 72 °C for 2 min. The cycle was repeated 30 times and the reaction mixture was incubated at 72 °C for additional 7 min. to allow for primer extension. The PCR amplified product was separated from the primers on a 1% agarose gel. A well was carved ahead of the required fragment and filled with 15% PEG-T AE solution (Zhen and Swank, 1993). The DNA was electro-eluted in 15% PEG-TAE solution, and purified further by phenoVchloroform extraction and ethanol precipitation.

A standard PCR was set up as described below.

Polymerase Chain Reaction

L-[3,4,5-3H (N)]-Ieucine (143Cilmmol), [35S]-dATPaS, 1251-Na (350mCilml) were obtained from Amersham (England, UK).

Radioisotopes