Western blot handbook
Recommended loading controls
Running the SDS-PAGE gel
Transferring protein from gel to membrane
Immunoblotting and detection
Membrane stripping and reprobing
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It may be desirable to store a western blot for future analysis or reprobing. An advantage of PVDF membranes over nitrocellulose is the stability of PVDF. PVDF is a chemically resistant polymer that has excellent long term stability. Therefore, it is possible to store a PVDF membrane and preserve sample proteins for extended periods of time by following the protocol below.
Storing a blot protocol
- Sandwich the dry PVDF blot between two clean sheets of Whatman 3MM paper.
- Place the sandwich between two sheets of card stock or thin cardboard.
- Use paperclips to clip the stack together on the edges.
- Place the stack in a plastic bag and seal the plastic bag closed.
- Store the blot at 4 ˚C for up to 2 weeks, -20 ˚C for up to 2 months, or -70 ˚C for longer storage.
- TIP: Thaw frozen blots to room temperature before removal from the plastic bag because frozen blots are prone to breakage.
- TIP: Blots can also be stored wet at 4 ˚C, but sodium azide should be added to prevent bacterial growth. As sodium azide inhibits HRP activity, it should be thoroughly washed out of a blot prior to use.
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