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OUTLINE

Western blot is a wide used technique to identify a target protein/s for the certain antibody.

PROTOCOL
  1. Prepare platelets.
  2. Lyse washed platelets (109 platelets/ml) with 1ml of ice-cold complete lysis buffer. Let to stay for 30min on ice.
  3. Remove debris by centrifugation at 100,000 g for 15 min.
  4. Measure the total protein concentration by a Lowry assay (Lowry et al, 1951).[  read more  ]
  5. 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.
  6. Prepare and load the running gel. Afterwards, prepare and load stacking gel. Use a comb to form loading "wells" for samples of proteins.
  7. Place the gel into the current field (20 mA per stacking gel, 30 mA per running gel)
  8. Transfer proteins to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech, England) (200 mA-1 A, 2 hours).
  9. Sink in Ponceau S solution. Get rid of the Ponceau S solution by washing with ddH2O.
  10. Block membranes for 1 hour at room temperature with PBS containing 5% dried nonfat milk.
  11. Wash 1x15 min and 2x5 min in PBS containing 0.05% Tween-20
  12. Incubate overnight at 4ƒC with the first Ab in PBS containing 5% dried nonfat milk.
  13. Wash 1x15 min and 2x5 min  in PBS containing 0.05% Tween-20
  14. Incubate for 1 hour at room temperature with the right dilution of revealing antibody conjugated to horse-radish peroxidase.
  15. Wash 1x15 min and 2x5 min  in PBS containing 0.05% Tween-20
  16. 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
  1. 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.
  2. 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
  3. 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
  4. upper TRIS, 500 ml, water sol., pH 6.8 , store at 4ƒC, use at RT = tris-HCl, 30.3 g (0.5 M) + SDS, 2 g (0.4% final)
  5. lower TRIS, 500 ml, water sol., pH 8.8, store at 4ƒC, use at RT = tris, 90.85 g (1.5 M) + SDS, 2 g (0.4 % final)
  6. sample buffer, 50 ml, store at -20ƒC, 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
  7. running buffer (tank), x10, 1 L, water solution, do not correct pH, store at 4ƒC, use at RT = tris base, 30.3 g (0.25 M) + glycine, 144.1 g (1.92 M) + SDS, 10 g (1% final).
  8. running buffer x1, 1 L = 100 ml of running buffer x 10 + ddH2O, 900 ml
  9. transfer buffer, x10, 1 L, water solution, pH 8.3 (do not correct pH), store at 4ƒC, use at RT = tris base, 14.54 g (120 mM) + glycine, 72 g (960 mM)
  10. transfer buffer, x1, 1 L = transfer buffer, x10, 100 ml (10% final)+ methanol (extra), 200 ml (20% final) + ddH2O, 700 ml
  11. Ponceau S, 1 L, keep at RT = Ponceau S, 1g + acetic acid (glacial), 50 ml + ddH2O, up to 1 L
  12. 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
  13. Decoloration solution, keep at RT = CH3OH, 500 ml + Acetic Acid (glacial), 100 ml + ddH2O, up to 1 L
  14. PBS-tween 20 = 2 L of PBS + 1 ml of tween 20
ADDITIONAL INFO
  1. 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.
  2. [  read more  ] about gel electrophoresis of proteins and staining prcedure from Amersham Biosciences.
SCHEME
western blot
[  enlarge  ]

REFERENCES
  1. 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.
  2. Schägger, H., and von Jagow, G. Anal. Biochem. 166, 368ñ379 (1987). Westermeier, R. Electrophoresis in Practice 2nd ed. VCH, Weinheim, Germany (1997).
  3. 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. 1ñ52 (1998).
created by Andrei Musaji          ::          updated : 2004