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Stripping and Reusing Western Blots
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.
Reagents:
TBST:
0.3 ml of Tween-20
30 ml of 10X TBS
Add ddH2O to a total volume of 300 ml
TBS (10X):
For 1 liter:
200 ml 1M Tris-HCl, pH 7.6 (final concentrtion, 200 mM)
275 ml 5M NaCl (final concentraion: 1.375 M)
Store at room temperature.
SDS 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
Procedure
A. Method 1 for Stripping Westerns
1. Place the Western blot to be reprobed in a volume of SDS 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 (At this point, the blot may be stored at 4°C until needed).
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 At this point, the blot may be stored at 4°C until needed).
6. Reblock the membrane with TBSTM before reprobing with the second or subsequent antibody .
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