WesternBlotting

实验概要

Western Blot (AP)

主要试剂

1. Membrane Blocking buffer:5% Milk   0.05% of Tween 20 PBS
2. antibody dilution buffer: PBS 0.05% of Tween20 1.0% Milk(or BSA) 
3. washing buffer: PBS 0.05% of Tween20

实验步骤

Western Blot (AP)

1. Membrane Block

     5% Milk   0.05% of Tween 20, Sharking for 2hr

2. Wash the membrane 3X with PBS(0.05% of Tween 20), 10min each

3. Primary antibody

      Add primary antibody (antibody mix with PBS 0.05% of Tween20 1.0% Milk(or BSA) with proper dilution), 4◦C, O/N.

4. Wash the membrane 3X with PBS(0.05% of Tween 20), 10min each

5. Secondary antibody

      Add second antibody (antibody mix with PBS 0.05% of Tween 20, for AP) RT,

       1 hr.

6. Wash the membrane 3X with PBS(0.05% of Tween 20), 10min each

7.  Exposure the membrane in AP buffer.