Biological Materials GST antibodies

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2.1.1.2. Purified rabbit anti-GST:

GST antibodies had been previously prepared in Dr.Karim's lab by immunizing white New Zealand strain rabbits that were purchased from Nile Company for Drugs Amerya, Cairo, with purified GST antigen using Freund's complete adjuvant containing heat-killed Mycobacterium tuberculosis.

2.1.1.3. Bacterial Strains:

The bacterial strains used in this study, and their general characteristics are outlined in (Table 4).

Table 4: Bacterial Strains

Strain

Genotype

Remarks

Escherichia coli

Top10 cells

(Invitrogen)

F-mcrA(mrr- hsdRMS-mcrBC) ِ 80 lacZ M15 lacX74 recA1 araD139 (araleu) 7697 galU galK rpsL (StrR) endA1 nupG

TOP10 E. coli are provided at a transformation efficiency of 1 x 109 cfu/µg supercoiled DNA. hsdR for efficient transformation of unmethylated DNA from PCR amplification. mcr for efficient transformation of methylated DNA from genomic preparations .lacZM15 for blue/white color screening of recombinant clones. endA1 for better results in applications, due to the elimination of non-specific digestion by Endonuclease I. recA1 for reduced occurrence of non-specific recombination in cloned DNA (Grant, 1990).

2.1.2. Vectors and Primers:

2.1.2.1. Vectors:

i) TA Cloning Kit was purchased from Invitrogen.

ii) pGEX-4T-1 expression vector was purchased from Pharmacia LKB corp.

iii) Plasmids with cloned HCV type 4a genome, isolate ED43 (Chamberlain et al., 1997) were provided by Dr. Richard Elliott (institute of virology, university of Glasgow).

2.1.2.2. Oligonucleotide primers:

Oligonucleotide primers for PCR amplification were kindly provided by Dr. Edward Niles, Buffalo University, N.Y., U.S.A. Primers span the HCV genome sequences plus BamHI or SalI cut site for forward and reverse primers, respectively (Table 5). Those terminal cut sites were designed for in frame directional cloning into the pGEX-4T-1 expression vector.

Table 5: Oligonucleotide primers

*Primer name and positions on HCV sequence

Sequence

1 (+) 3601

GGG GGA TCC TTG GTG GGG TGG CCA GC

2 (-) 4200

GGG GTC GAC CTA TTC GCT GCA ACC TCC ATC

3 (+) 4801

GGG GGA TCC CCA TCA GGC ATG TTT GAC

4 (-) 5400

GGG GTC GAC CTA TTC GTC GAA CTG TTG GTA

5 (+) 6001

GGG GGA TCC CCG GGC GAA GGG GCC

6 (-) 6600

GGG GTC GAC CTA TTG TGT TAC CCC GGT GAC

7 (+) 3301

GGG GGA TCC AAT GAG ATC TTG CTC GGA C

8 (-) 3900

GGG GTC GAC CTA TGA TCT CAT GGT AGT CTC

*Based on Hepatitis C Virus genotype 4a isolate ED43 nucleotide sequence EMBL accesion number Y11604.

 (+) is the Forward primer

 (- ) is the Reverse primer

 Underlined sequence represents restriction recognition sites

 The sequence GGG was added at the 5' and 3' ends of primers to enhance restriction enzymes to digest exactly at their proper cut sites and avoid flanking.

2.1.3. Culture Media:

2.1.3.1. LB Liquid medium:

To 900 ml of distilled water, add:

Bacto-tryptone (Difco) 10.0 g

Bacto-yeast extract (Difco) 5.0 g

NaCl (Sigma) 10.0 g

Distilled water 900.0 L

The pH was adjusted to 7.0 with 5N NaOH, then the volume of the solution was completed to 1 liter, and the medium was sterilized by autoclaving for 20 minutes at 121°C. For preparing solid medium, the same recipe of 2xYT-medium was prepared, followed by the addition of bacto-agar (Difco) up to 1.5% (w/v), sterilized by autoclaving for 20 minutes at 121°C then allowed to cool to 50°C before pouring into petridishes. After autoclaving, filtered sterilized ampicillin (100 mg/ml) or Kanamycin (50mg/ml) was added to a final concentration (100 g/ml)or (50g/ml) respectively.

2.1.3.2. Ampicillin (Sigma) (100 mg/ml):

Ampicillin 1.0 g

Distilled water 10.0 ml

The solution was sterilized by filtration through a 0.2m filter, then divided into 0.5 ml aliquots and stored at -20°C.

2.1.3.3 Kanamycin (sigma) ( 50mg/ml)

Kanamycin 0.5g

Distilled water 10.0ml

The solution was sterilized by filtration through a 0.2m filter, then divided into 0.5 ml aliquots and stored at -20°C.

2.1.4. Kits:

TA Cloning Kit, was purchased from Invitrogen, (Buffalo, NY, USA).

2.1.5. Enzymes:

 BamHI, was purchased from Promega Corporation (Madison, WI, USA).

 T4 DNA ligase was purchased from Fermentas (USA)

 SalI and Taq DNA polymerase were purchased from Fermentas (USA)

2.1.6. Reagents for antibody and protein analysis:

 Protein molecular weight marker for SDS-PAGE was purchased from Fermentas (USA)

2.1.7. Chemicals:

Most chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and from International Biotechnologies, Inc. (IBI; a subsidiary of Eastman Kodak Company, New Haven, CT, USA).

 DNA molecular weight marker 1kb ladder was purchased from Fermentas (USA)

 Agarose, molecular biology certified, was purchased from International Biotechnologies Inc. (IBI).

 dNTPs (dATP, dCTP, dGTP and dTTP) were purchased from Fermentas (USA)

2.1.8. Supplies:

 Sony films, type I, Normal UPP110 were purchased from Sony Corporation (Tokyo, Japan).

 Nalgene disposable filterware, 0.45 and 0.2 µm pore size, were purchased from Nalge Co., a subsidiary of Sybron Corp. (Rochester, NY, USA).

 Glass wool was provided by VACSERA.

2.1.9. Reagents and Solutions (Sambrook et al., 1989):

 30% Acrylamide (Acrylamide stock solution for protein electrophoresis):

Acrylamide 29.0 g

N, N'-methylene-bis-acrylamide 1.0 g

Distilled water 60.0 ml

The pH was adjusted to 7.0, then the volume of the solution was completed to 100 ml, and sterilized by filtration through a Nalgene filter (0.45µm pore size). The acrylamide was stored in a dark bottle at room temperature.

 10% Ammonium persulfate:

Ammonium persulfate 0.1 g

Distilled water to 1.0 ml

The solution was divided to aliquots and stored at 4°C for up to several weeks.

 Chloroform-iso-amyl alcohol (Chisam):

Chloroform 24.0 volumes

Iso-amyl alcohol 1.0 volume

 Coomassie destaining solution:

Methanol 45.0 ml

Distilled water 45.0 ml

Glacial acetic acid 10.0 ml

 Coomassie staining solution (SDS-PAGE staining solution):

Coomassie Brilliant Blue R250 0.25 g

Methanol 45.00 ml

Distilled water 45.00 ml

Glacial acetic acid 10.00 ml

The solution was mixed well and filtered through a Whatman No. 1 filter and stored in an aluminum foil wrapped bottle at room temperature.

 Ethylene diamine tetra acetic acid (EDTA), 0.5M, pH 8.0:

EDTA 18.61 g

Distilled water 80.00 ml

The pH was adjusted to 8.0 with 10N NaOH, the volume was completed to 100ml, dispensed into aliquots and sterilized by autoclaving.

 Ethidium bromide, 10 mg/ml:

Ethidium bromide 0.1 g

Distilled water 10.0 ml

The solution was dissolved by stirring on magnetic stirrer for several hours, and stored at room temperature in an aluminum foil wrapped tube.

 Isopropylthio--D-galactoside (IPTG), (M.W = 238.8):

IPTG 1.0 g

Distilled water to 5.0 ml

The solution was sterilized by filtration through a 0.2 m filter, dispensed into 1ml aliquots and stored at -20°C.

 Phenol: (equilibrated to pH >7.8):

The phenol was melted at 68°C then hydroxyquinoline was added as an antioxidant to a final concentration of 0.1 %. Equal volume of 0.5 MTris.Cl pH 8.0 was added to the liquified phenol and stirred for 15 minutes at room temperature. After the two phases had separated, as much as possible of the aqueous (upper) phase was aspirated off. Equal volume of 0.1M Tris.Cl pH 8.0 was added to the phenol, stirred, the phases were left to separate, and the upper aqueous phase was aspirated off as before. This step was repeated for several times until pH of phenolic phase was >7.8. The final aqueous phase was discarded and 0.1M Tris.Cl pH 8.0 was added. The phenol was then stored in this form in a light-tight bottle at 4°C for periods of up to 1 month.

 Phenol-Chloroform:

Equilibrated phenol 1 volume

Chloroform 1 volume

Stored under 0.01M Tris.Cl pH 7.6 at 4°C in dark glass bottle.

 Potassium acetate buffer (5M):

Potassium acetate 49.1g

Distilled water 80.0 ml

 Ribonuclease, pancreatic (RNase A), 10 mg/ml:

RNase A 10 mg

Distilled water 1 ml

The solution was boiled for 15 minutes, then cooled to room temperature before dispensing into 100 μl aliquots and stored at -20°C.

 3M Sodium acetate, pH 5.2:

Sodium acetate 20.4 g

Distilled water 40.0 ml

The pH was adjusted to 5.2 with glacial acetic acid then the volume was completed to 50 ml with distilled water and dispensed into aliquots and sterilized by autoclaving.

 10% Sodium dodecyl (lauryl) sulfate (SDS):

SDS 5 g

Distilled water 40 ml

The solution was heated to 68°C, pH was adjusted to 7.2 by adding a few drops of concentrated HCl and the volume was adjusted to 50 ml with distilled water.

 10N Sodium hydroxide:

Sodium hydroxide 20 g

Distilled water to 50 ml

 Solution I: (For alkaline lysis of plasmid DNA mini preparation):

glucose 0.9 g (50mM final)

1MTris-HCl, pH 8.0 2.5 ml (25 mM Final)

0.5M EDTA, pH 8.0 2.0 ml (10mM final)

Distilled water to 100.0 ml.

The solution was autoclaved for 20 minutes at121°C, and stored at 4°C.

 Solution II: (For alkaline lysis of plasmid DNA mini preparation):

10 N NaOH 2 ml (0.2 N final)

10% SDS 10 ml (1% final)

Distilled water to 100 ml

 Solution III: (For alkaline lysis of plasmid DNA mini preparation):

5M Potassium Acetate 60.0 ml

Glacial Acetic Acid 11.5 ml

Distilled water 28.5 ml

The resulting solution is 3M with respect to potassium and 5M with respect to acetate.

 1M Tris:

Tris base 121.1 g

Distilled water 800.0 ml

The pH was adjusted with concentrated HCl to the desired value then the volume was completed to 1L, dispensed into aliquots and sterilized by autoclaving.

 X-gal (5-Bromo-4-chloro-3-indolyl--D-galactoside), 20 mg/ml:

X-gal 20 mg

Dimethylformamide 1ml

Stored in aluminum foil wrapped tubes at -20°C

.

2.1.10. Buffers:

 Agarose-DNA loading buffer: (6x stock):

Bromophenol blue 25 mg (0.25% final)

Sucrose 4 g (40% final)

Distilled water to 10 ml

The solution was stored in aluminum foil wrapped tubes at 4°C.

 Saline-Tris-EDTA Buffer (STE Buffer):

5M NaCl 10 ml ( 0.1 M final)

1M Tris-HCl pH8.0 5 ml (10.0 mM final)

0.5M EDTA pH8.0 1 ml ( 1.0 mM final)

Distilled water to 500 ml.

 SDS gel-electrophoresis buffer; pH 8.3 (5x stock):

Tris base 15.1 g (0.125M final)

Glycine 94.0 g (1.25M final)

10% SDS 50.0 ml

Distilled water to 1.0 L

The stock SDS gel-electrophoresis buffer was diluted 5 times to give a 1x working solution.

 SDS gel-loading buffer (2 x stock):

1M Tris-HCl; pH 6.8 1.0ml (100 mM final)

10% SDS 4.0 ml (4% final)

Glycerol 2.0 ml (20% final)

-Mercaptoethanol 1.0 ml (1.44M final)

Bromophenol blue 20.0 mg

Distilled water to 10.0 ml

The solution was dispensed into aliquots and stored at -20°C.

 Tris-Acetate-EDTA (TAE; 50x stock):

Tris base 242.0 g

Glacial acetic acid 57.1 ml

0.5M EDTA; pH 8.0 100.0 ml

Distilled water to 1L

The stock Tris-Acetate-EDTA buffer was diluted 50 times to give a 1x working solution

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