Home | Achievement | Programmes | Projects | Experts | Staffs | Publications | Journals |
Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |

MOLECULAR BIOLOGY: WORKING WITH DNA

DETECTION: MEMBRANE TRANSFER

Stripping and Reusing Western Blots

Stripping and Reusing Western Blots Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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 (see Protocol ID#1146). 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 (see Protocol ID#1146).
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
SDS Sodium Chloride Glycine HCl 2-Mercaptoethanol Tris HCl Hydrochloric Acid Tris Tween-20
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. At this point, the blot may be stored at 4°C until needed.