Protocols

Stripping and Reusing Western Blots

Posted on Sunday, October 19, 2003

Description
A single Western blot is often probed serially with multiple antibodies. In order to avoid an interfering signal from a previous experiment, the Western blot can be treated to remove antibodies from the membrane. This protocol describes two methods of removing a previous antibody on the Western blot before proceeding with a subsequent antibody.

Procedure
A. Method 1 for Stripping Westerns

1. Place the Western blot to be reprobed in a volume of Stripping Buffer sufficient enough to cover the membrane completely and allow free movement in the container.

2. Incubate in a sealed plastic container at 50°C for 30 min in a shaking water bath.

3. Rinse the membrane with 1X TBST (see Hint #1).

4. Reblock the membrane with TBSTM before reprobing with the second antibody.

B. Method 2 for Stripping Westerns

1. Place the Western blot to be reprobed in a volume of Glycine Stripping Buffer sufficient enough to cover the membrane completely and allow free movement of the membrane in the container.

2. Incubate the blot in a sealed plastic container at 60°C for 60 min in a shaking water bath.

3. Remove the solution from the blot. Replace it with fresh, prewarmed Glycine Stripping Buffer.

4. Incubate again in a sealed plastic container at 60°C for 60 min in a shaking water bath.

5. Rinse the blot with 1X TBST (see Hint #1).

6. Reblock the membrane with TBSTM before reprobing with the second or subsequent antibody .


Recipes
TBST 0.3 ml of Tween-20
Add ddH2O to a total volume of 300 ml
30 ml of 10X TBS


TBS (10X) 200 mM Tris, pH 7.6
Store at room temperature
pH to 7.6 with HCl
1.37 M NaCl


Stripping Solution 2% (w/v) SDS
100 mM 2-Mercaptoethanol
62.5 mM Tris-Cl, pH 6.7


Glycine Stripping Buffer 0.2 M Glycine-HCl, pH2.5
0.05% Tween-20
100 mM 2-Mercaptoethanol



Supplies


Tips
1. At this point, the blot may be stored at 4°C until needed.

Submitted by: manny440