Western blotting – probing the membrane with antibodies

Steps

1. Block the membrane at 4°C overnight or for 30 minutes at room temperature on rocker. (Make sure the side of the membrane that was facing the gel is facing up). A variety of blocking buffers can be used. A simple option includes 3-5% skim milk powder in TBST.

2. Dilute primary antibodies 1 in 500 - 2000 (every antibody is different) in TBS and incubate with the membrane for 2 hours at room temperature or 4°C overnight gently on a rocker or orbital shaker.

3. Wash the membrane 4 times in plain TBST.

4. Dilute secondary antibodies 1 in 5000 in TBS and incubate with the membrane for 1 hour at room temperature.

5. Wash 4 times with TBST (4 minutes per wash). The membrane is now ready for imaging.

TBS (Tris buffered saline)
20mM Tris, 140mM NaCl, deionised water

TBST
TBS with 0.05% Tween 20