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How to Get Perfect Protein Transfer in Western Blotting

Accurate transfer of proteins from the SDS-PAGE gel to your membrane is an important step in Western blotting.  However, optimizing transfer times is hit-or-miss, and it can take several tries to get a publication-worthy image.  Here are a few hints on how to ensure that your transfer is accurate and complete:

    • Always include a pre-stained molecular weight marker.  A pre-stained marker not only allows you to monitor how far your gel has run; it is also very handy for detecting how much of your protein has transferred to the blotting membrane.  When you check your membrane after transfer, make sure that every band from the molecular marker has transferred clearly and completely to the membrane.
    • Add a second membrane to the transfer apparatus.  Small molecular weight proteins transfer readily, so much so that they may actually transfer through the membrane before the larger proteins have made it all the way across.  If you suspect that you are losing some of your smaller proteins due to this phenomenon, try adding a second membrane to your transfer cassette, and blotting both.  If you can detect lower molecular weight proteins on the second membrane, then the transfer reaction is going too long.
    • Stain your protein gel after transfer.  If your transfer doesn’t run long enough, you can be leaving protein behind in the gel.  The likelihood of this happening increases if you’re trying to transfer high molecular weight proteins, which are less mobile than smaller proteins.  To test whether you are getting complete and even transfer of all of your proteins, try staining the gel after transfer.  By using silver stain or coomassie stain, you should be able to detect any protein still remaining in the gel that didn’t make it onto the membrane.

  Source:  This excellent video by Agrisera, which has a lot of great tips on how to make beautiful western blots.

What are your helpful hints on how to optimize your transfer?

1 Comment

  1. 5 to 16 chars on August 17, 2011 at 11:15 am

    My understanding is that if smaller proteins are running through the membrane, then you need to transfer at a lower voltage, not for less time (assuming the time is correct for the larger molecules). The theory is that lower voltage will not allow the transferring proteins from becoming “unstuck” from the front face of the membrane.
    Also, most commercial sample buffers include Coomassie G250, which tracks the ion front when running the SDS-PAGE gel. Hence, you can just stop the electrophoresis run when the dye front reaches the bottom of the gel.

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