Immunodetection for Specific Proteins


This is the standard protocol I use for immunodetection after proteins are transferred to a PVDF membrane from a gel. The procedure is essentially a series of antibody cross-linking reactions and washes, concluding with a color reaction revealing where particular proteins are on the membrane. The theory behind the procedure can be found in introductory immunology textbooks. Generally, antibodies are raised in some animal (e.g. a rabbit) by injecting the target antigen (e.g. a known protein, such as human spectrin) into the animal. This is the "primary antibody" and is used to bind to specific proteins (if present on a PVDF membrane) from test samples. A "secondary antibody" is used to bind to the first. In the procedure I use, the second antibody is conjugated (i.e. covalently bound) to an enzyme which catalyses a color reaction. Thus, bands appear after a short while (15 to 60 min) anywhere the secondary antibody is bound, and this shows where particular proteins are on the PVDF membrane. Throughout the entire procedure (below), a "shaker" is used. The shaker is simply a small machine with a platform that rocks back and forth to move or "wash" liquid over the PVDF membrane. Depending on the experiment being carried out, individual lanes (which are cut from the blot) or the whole blot (ten lanes) can be stained. The volumes that I use for each step are (typically): 2 ml per lane, if separate lanes are being stained; 10 ml if the whole blot is being stained.

Immunodetection Protocol (Western blot)

  1. Prewet PVDF membrane in methanol
  2. Wash in PBS for 10 min on shaker
  3. 45 min with Blocking Buffer (shaking)
  4. 1 hour for primary antibody (shaking)
  5. Wash 3 x 10 min in 1/2 BB (1:1 v/v, PBS to Blocking Buffer) (shaking)
  6. 1 hour for secondary antibody (shaking)
  7. Wash 1 x 10 min in 1/2 Blocking Buffer (shaking)
  8. Wash 2 x 10 min in PBS (shaking)
  9. Stain in NBT/BCIP solution. Rinse in dH20 after 1 hour.

PBS (Phosphate Buffer Saline) pH 7.4
8g NaCl
0.2 g KCl 1.44 g Na2HPO4
0.24 g KH2PO4
add dH2O to 1 liter

Blocking Buffer (BB)
in PBS to 100 ml
5% of milk powder
0.1% Tween 20
0.03% NaN3

NBT/BCIP
200uL NBT/BCIP in 10 mL of buffer:
0.1 M Tris-HCl pH 9.5
0.05 M MgCl2
0.1 M NaCl
SITE MAP

EVOLUTION INDEX.

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