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.


  1. Graciela on April 13, 2017 at 11:52 am

    I have a question, and it would be nice some help because I don’t find anything like that on the problems on internet: I have transferred the gel which migrated well apparently seeeing the ladder (no smiles no bizarre things) and it didn’t tranferred AT ALL to the membrane, it didn’t wen’t to the papers in front or behind, and it is not neither in the gel, nor the ladder. I reapeat, nor the proteins, nor the ladder are transferred: the membrane is WHITE, plain WHITE, as well as everything in front and forward.
    The buffers were new, I controlled the Tº, the speed was 90V 1:40h,
    how is this possible? What troubles me more is that not even the ladder was transferred, LIKE no signal
    I’m going to stain the gel (see if “some proteins left”..), but I don’t want to charge more samples as they are precious and we’re running out.
    Any help or ideas are welcome
    Thanks in advance

    • Dr Amanda Welch on April 13, 2017 at 5:31 pm

      Have you tried running a different sample? What membrane did you use (nitrocellulose or PVDF)? It’s possible to transfer through the membrane, whatman paper, and to the sponge—I’ve done it, but it took a large amount of forgetfulness and some miscommunication.

  2. Emily Crow on March 14, 2012 at 7:29 am

    Good call on the Ponceau-S, poorpostdoc! Tried and true, and very simple.

    Do you use amido black to stain your membrane for your lab notebook?

    • poorpostdoc on March 16, 2012 at 6:33 am

      Yes, staining with Amido black for the lab notebook works really well. It also helps align the blot to the film at the end of the whole Western process.

  3. poorpostdoc on March 12, 2012 at 4:24 pm

    One of the most basic checks for protein transfer: Ponceau-S staining. There are several variations of the recipe out there, the most simple being 0.1% w/v Ponceau-S in 5% acetic acid. Agitate your blot in the stain for 2-5 minutes, rinse with distilled water. After you’ve seen your proteins have transferred, continue to rinse, or go straight into a blocking step. The dye will wash out completely.

    An extra tidbit: After you have done your Western blot visualization, stain the blot with amido black:

    Stain: 0.1% (w/v) amido black in 25% (v/v) isopropanol, 10% (v/v) acetic acid
    Destain: 25% (v/v) isopropanol, 10% (v/v) acetic acid

    Agitate your blot in the stain for 10 minutes, rinse with distilled water. Place the blot in destain solution and agitate for 5 to 10 minutes to remove excess stain. I usually repeat the destain process.

    There is an article that suggests the use of reversible amido black staining for nitrocellulose: BLOEMENDAL, H., and SATIJN, M. (1995). Simple Reversible Staining of Proteins Transferred From Polyacrylamide Gels Onto Nitrocellulose Membranes. Anal Chim Acta 300, 1–3.

    • poorpostdoc on March 12, 2012 at 4:25 pm

      And by “After you have done your Western blot visualization” I mean – after you have your film or phosphorimager image or whatever your lab is using.

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