Dot Blot Protocol

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Dot Blot Protocol


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.

A) 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.)

B) Procedure
1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region you are going to blot (see
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 10cm Petri Dish for reaction
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 manufacturers
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.