OUTLINE
Western blot is a wide used technique to identify a target protein/s for the certain antibody.
PROTOCOL
Western blot is a wide used technique to identify a target protein/s for the certain antibody.
PROTOCOL
- Prepare platelets.
- Lyse washed platelets (109 platelets/ml) with 1ml of ice-cold complete lysis buffer. Let to stay for 30min on ice.
- Remove debris by centrifugation at 100,000 g for 15 min.
- Measure the total protein concentration by a Lowry assay (Lowry et al, 1951).[ read more ]
- Add either reducing or non-reducing sample buffer to a protein probe. Mix well. Boil for 5 min. Centrifuge for 10 min. at 14 000 rpm (100,000g) (ultracentrifuge) if needed.
- Prepare and load the running gel. Afterwards, prepare and load stacking gel. Use a comb to form loading "wells" for samples of proteins.
- Place the gel into the current field (20 mA per stacking gel, 30 mA per running gel)
- Transfer proteins to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech, England) (200 mA-1 A, 2 hours).
- Sink in Ponceau S solution. Get rid of the Ponceau S solution by washing with ddH2O.
- Block membranes for 1 hour at room temperature with PBS containing 5% dried nonfat milk.
- Wash 1x15 min and 2x5 min in PBS containing 0.05% Tween-20
- Incubate overnight at 4ѓC with the first Ab in PBS containing 5% dried nonfat milk.
- Wash 1x15 min and 2x5 min in PBS containing 0.05% Tween-20
- Incubate for 1 hour at room temperature with the right dilution of revealing antibody conjugated to horse-radish peroxidase.
- Wash 1x15 min and 2x5 min in PBS containing 0.05% Tween-20
- Reveal bound antibodies with ECL western blotting detection reagents (Amersham Pharmacia Biotech, England) according to the manufacturer's instructions. Visualize the chemoluminiscence with the X-Ray film.
SOLUTIONS
- complete lysis buffer = 50 mM Tris HCl, 150 mM NaCl, 1mM EDTA, 1% triton X-100, 10% glycerol, 1 mM NaF, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, pH 7.4.
- running gel (lower), V=20 ml
substance/gel conc./ % 5% 7% 10% acrylamid/bis-, 40% 2.5 ml 3.52 ml 5 ml ddH2O 11.7 ml 10.68 ml 9.2 ml lower TRIS 5 ml 5 ml 5 ml glycerol 50%, wtare sol. 0.4 ml 0.4 ml 0.4 ml Temed 25 µl 25 µl 25 µl Ammonium persulfate, 1% 0.8 ml 0.8 ml 0.8 ml - stacking gel (upper), 3%
substance amount acrylamid/bis-, 40% 1.2 ml ddH2O 10 ml upper TRIS 4 ml TEMED 16 µl Ammonium persulfate, 1% 0.8 ml - upper TRIS, 500 ml, water sol., pH 6.8 , store at 4ѓC, use at RT = tris-HCl, 30.3 g (0.5 M) + SDS, 2 g (0.4% final)
- lower TRIS, 500 ml, water sol., pH 8.8, store at 4ѓC, use at RT = tris, 90.85 g (1.5 M) + SDS, 2 g (0.4 % final)
- sample buffer, 50 ml, store at -20ѓC, use at RT
substance/ conc., times x2 x3 glycerol 10 ml 15 ml SDS 3 g 4.5 g upper TRIS 12.5 ml 18.75 ml bromphenol blue 10-40 mg 50 mg ddH2O up to 50 ml up to 50 ml b-mercaptoethanol
(only for reducing-conditions)5 ml 7.5 ml - running buffer (tank), x10, 1 L, water solution, do not correct pH, store at 4ѓC, use at RT = tris base, 30.3 g (0.25 M) + glycine, 144.1 g (1.92 M) + SDS, 10 g (1% final).
- running buffer x1, 1 L = 100 ml of running buffer x 10 + ddH2O, 900 ml
- transfer buffer, x10, 1 L, water solution, pH 8.3 (do not correct pH), store at 4ѓC, use at RT = tris base, 14.54 g (120 mM) + glycine, 72 g (960 mM)
- transfer buffer, x1, 1 L = transfer buffer, x10, 100 ml (10% final)+ methanol (extra), 200 ml (20% final) + ddH2O, 700 ml
- Ponceau S, 1 L, keep at RT = Ponceau S, 1g + acetic acid (glacial), 50 ml + ddH2O, up to 1 L
- Coomassie dye, 1 L , keep at RT , dark = Coomassie Blue R-250; 1g + C2H5OH (extra), 400 ml + Acetic Acid (glacial), 100 ml + ddH2O, up to 1L
- Decoloration solution, keep at RT = CH3OH, 500 ml + Acetic Acid (glacial), 100 ml + ddH2O, up to 1 L
- PBS-tween 20 = 2 L of PBS + 1 ml of tween 20
ADDITIONAL INFO
- When gel is run in duplicate, the second gel could be used for Coomassie staining for 5-10 min in Coomoasie solution followed by decolorant solution incubation for 2 min.
- [ read more ] about gel electrophoresis of proteins and staining prcedure from Amersham Biosciences.
SCHEME
REFERENCES
REFERENCES
- Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with folin phenol reagent. J. Biol. Chem. 1951. 193: 265-275.
- Schägger, H., and von Jagow, G. Anal. Biochem. 166, 368с379 (1987). Westermeier, R. Electrophoresis in Practice 2nd ed. VCH, Weinheim, Germany (1997).
- Shi, Q. One-dimensional polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach 3rd ed. (Hames, B. D., ed.), Oxford University Press, New York, pp. 1с52 (1998).
