Dotblotprotocol

實驗概要

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.

主要試劑

1. TBS
20 mM Tris-HCl
150 mM NaCl
pH 7.5

2. TBS-T
0.05% Tween20 in TBS

3. BSA/TBS-T
0.1% BSA in TBS-T

主要設備

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

實驗步驟

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.