Dot blot protocol

General dot blot procedure

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A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate.

Concentration of proteins in crude preparations (such as culture supernatant) can be estimated semiquantitatively by using dot blot method if you have both purified protein and specific antibody against it.

Reagents

TBS:
20 mM Tris-HCl
150 mM NaCl
pH 7.5

TBS-T:
0.05% Tween20 in TBS

BSA/TBS-T:
0.1% BSA in TBS-T

Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.)

Procedure

 

  1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region you are going to blot (see below).
  2. Using narrow-mouth pipet tip, spot 2 μl of samples onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 3-4 mm diam.) by applying it slowly.
  3. Let the membrane dry.
  4. Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). Use 10 cm Petri Dish for reaction chamber.
  5. Incubate with primary antibody (0.1-10 μg/ml for purified antibody, 1:1000 to 1:100000 dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 30 min at RT.
  6. Wash three times with TBS-T (3 x 5 min).
  7. Incubate with secondary antibody conjugated with HRP (for optimum dilution, follow the manufacturer’s recommendation) for 30 min at RT.
  8. Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with TBS (5 min).
  9. Incubate with ECL reagent for 1 min, cover with Saran-wrap (remove excessive solution from the surface), and expose X-ray film in the dark room. Try several different lengths of exposure.
  10. Compare the signal from your unknown sample to that of standard and estimate the concentration.