How many times can I strip my blot using Restore or Restore Plus Western Blot Stripping Buffers ?
The stability of the attached (transferred and bound) protein will determine the number of times the membrane can be successfully re-probed after stripping. The protein may withstand stripping as many as four times or as few as one time.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What is the proper wash buffer to use after stripping with Restore or Restore Plus Western Blot Stripping buffers?
Wash the membrane with the same buffer as was used between antibody probing steps during the Western blotting procedure.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Can I strip membranes with Restore or Restore Plus Western Blot Stripping Buffer and reprobe for subsequent detection with fluorescent probes?
No. Although the fluorescent antibodies, like other antibodies, can be stripped with Restore Buffers, stripped membranes typically produce unacceptable background for subsequent fluorescent detection methods. We recommend Restore Fluorescent Western Blot Stripping Buffer, Cat. No. 62299 (20 mL) and Cat. No. 62300 (100 mL).
Note: Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane (Cat. No. 22860) only.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Will the Restore and Restore Plus Western Blot Stripping Buffers remove precipitating substrates?
No. The antibodies are removed but the substrate leaves a permanent precipitate on the membrane that cannot be removed. Restore and Restore Plus Buffers are designed for procedures using chemiluminescent substrates. Please note that this is not compatible with fluorescence supstrates as it will result in increased background. For fluorescent substrates please use our Restore Fluorescent Western Blot Stripping Buffer. Please also see Tech Tip: Strip and Reprobe Western Blots (https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/strip-reprobe-western-blots-tech-tip.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
If I want to strip and re-probe a nitrocellulose membrane, what procedure do you recommend?
Thermo Fisher Scientific sells 4 ready-to-use buffers specifically intended for stripping Western blots prior to re-probing. Three of them can be used with both nitocellulose and PVDF membranes. We have Restore Western Blot Stripping Buffer (Cat. No. 21059), a trial size of this product (Cat. No. 21062), a product called Restore PLUS (Cat. No. 46428), which is formulated to strip off antibodies that are difficult to remove, and Restore Fluorescent Western Blot Stripping Buffer (Cat. No. 62299). The latter is used to strip fluorophore-labeled antibodies from PVDF membranes only.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.