Restore™ Fluorescent Western Blot Stripping Buffer

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.

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Wash the membrane with the same buffer as was used between antibody probing steps during the Western blotting procedure.

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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.

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.

For nitrocellulose or PVDF membrane following Western blot detection using a chemiluminescent or fluorescent substrate system: Following transfer, air dry the membrane and place in an envelope, preferably on top of a supported surface to keep the membrane flat. The blot can be stored indefinitely at -80 degrees C. When ready to reprobe, prewet the PVDF blot with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with blocking step.

FOR STRIPPING/REPROBING OF MEMBRANES: Harsh protocol (see NOTE below for modifications)

1) Submerge the membrane in stripping buffer (100 mM BME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubate at 50 degrees C for 30 min with occasional agitation. If more stringent conditions necessary, incubate at 70 degrees C.

2) Wash 2 x 10 min in TBS-T/PBS-T at room temperature.

3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hr at room temperature.

4) Immunodetection

NOTE: Often you don't need such harsh conditions to remove antibodies from their proteins. The stringency of one or several of the variables can be decreased: lower the temperature, decrease the time, less BME, less SDS, etc. An especially mild but still often effective stripping protocol is lower pH incubation. Example: pH 2.0 Tris 50-100 mM, 30-60 min incubation (you may do two incubations if you wish). Then rinse and block as usual. If you do not wish to re-use the membrane immediately after stripping, you can store the membrane in plastic wrap (wet, you do not want it to dry out). Another simple, mild stripping buffer is 0.1 M glycine•HCl (pH 2.5-3.0), incubation 30 min to 2 hrs room temperature or 37 degrees C, depending on the antibody.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.