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How to Optimize Your Western Blot Transfers

How to Optimize Your Western Blot Transfers

So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel.  Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot.

Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how long you need to run the transfer in order to get complete transfer of all your proteins.  Instead, you will need to optimize the transfer time and conditions for yourself, based on the equipment you use and the proteins you are working with.

So how do you know when your transfer is complete?  Here are a few good controls you can use to monitor transfer efficiency:

Look for complete transfer of the molecular weight ladder

Use a pre-stained protein ladder to track transfer of proteins from a gel to a membrane.  The rest of the proteins on your gel will of course be invisible, but you can easily see if the brightly colored ladder bands are still in the gel, or have transferred completely to the membrane.  Keep in mind that proteins of different sizes will migrate at different speeds: there is no magic moment at which all the proteins jump from the gel to the membrane.  Instead, you are aiming for a point at which most of the larger proteins have migrated to the membrane, and most of the smaller proteins are still on the membrane but not through it.  If you use a ladder where each band is stained a different color, this can help you keep track of the transfer efficiency of differently sized proteins.  Remember, if you’ve opened your transfer cassette to “peek” at the ladder, this will introduce air bubbles to the transfer stack – so you probably want to use this technique to optimize transfer times when you’re not running important samples.

After transfer, stain the SDS-PAGE gel with Coommassie blue

Another simple way to monitor the efficiency of protein transfer is to stain the gel with Coommassie after your transfer is complete.  This is a quick and easy procedure that will give you a good idea of how much protein is still in the gel: if the gel is mostly clear, than your transfer was successful, but if most of your gel shows blue bands, then your transfer needs to run longer.  Again, this is a “post-hoc” check for transfer efficiency: once you’ve stained the gel, you won’t be able to set it up for a second transfer.  Instead, you’ll need to run a second gel and do the transfer again using the adjusted conditions.  So remember to do this test before you run your precious samples!

Transfer your gel onto two membranes

The two methods described above are most useful for determining whether the transfer duration was too short.  To test is your transfer is too long, try inserting two membranes into your transfer stack, one right behind the other.  Then, go ahead and blot both membranes (be sure to label which was farthest away from the gel!).  If your transfer conditions are good, then the majority of your protein will be on the first membrane (closest to the gel).  If you are transferring too long, you will detect protein on the second membrane as well.  This is more likely to occur to the smallest proteins first so pay particular attention to where the lower molecular weight ladder bands should appear.  You can also try staining the second membrane with Coommassie blue, which will show you all of the proteins that have transferred to that layer, and not just your protein of interest.

For more information on these transfer controls, check out this video from Agrisera.

Originally published on March 12, 2012. Updated and revised on July 13, 2015.

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  1. Sepideh Esmaeily on March 1, 2018 at 8:40 am

    Thank you for your guidance.
    That is, the use of two membranes causes the protein samples transfer to membrane (PVDF). And that protein samples are transmitted on the first membrane or the second membrane?

  2. Bitesize Bio on November 17, 2015 at 7:15 am

    […] and tedious task to get a “presentable” gel for publication (especially when there are Western blots to follow)… but not if you know the […]

  3. Fuyao Li on September 5, 2015 at 1:12 pm

    Thanks for the suggestions! I am running Western Blots several times but no signals could be revealed after film development. I noticed that the pre-stained protein ladders were also transferred onto the filter papers during the semi-dry transfer. Is it possible that my protein samples were transferred onto the filter papers instead of the membrane? Thanks for the help!

    • Jordan Mohr on September 24, 2015 at 5:27 pm

      Chances are you either assembled the sandwich backwards, placed it in the transfer apparatus backwards, or attached the electrodes backwards to the apparatus. The protein is usually coated in negatively charged SDS (or maybe LDS), so it will migrate away from the black electrode to the red electrode during the transfer. Make sure you orient your sandwich with the gel towards the black electrode and the membrane towards the red electrode.

  4. Temitope Adelusi on July 27, 2015 at 8:17 am

    Thank you for the details You people are simply the best. I want more of these stuffs. Thank you very much.

    • Jennifer Redig on July 28, 2015 at 7:02 am

      i know I loved this site when I was in grad school. Glad you found it helpful. Anything in particular you would like to read about?

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