PVDF/Filter Paper Sandwich, 0.2 μm, 8.3 x 7.3 cm

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.

Drying the PVDF membrane reduces the background staining that can occur with wet membranes. A dry PVDF membrane is very hydrophobic and doesn't wet well, but the areas where proteins are bound are more easily saturated and stainable.

This will not work with nitrocellulose membranes, and will only work with PVDF membranes stained for a brief period; staining beyond the recommended time will only increase the background and reduce the detectability.

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After staining with SimplyBlue SafeStain, use deionized water for the less strongly retained protein bands on the PVDF membrane.

Increasing methanol or ethanol concentrations up to 70% should destain any remaining bands. You can leave the membrane in the destain indefinitely.

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We find that including ethanol in the transfer buffer is just as effective as including methanol. You may use either of these alcohols at an equivalent concentration.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking reagent 2 to 5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, thus reducing non-specific interactions with the primary antibody. We offer WesternBreeze Immunodetection kits with blocking reagents and Pierce Fast Western Blot kits that have been pre-optimized to give low-background blots. Other examples of blocking agents are nonfat dry milk, BSA, and casein, Starting Block, and SuperBlock.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.