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The Top 10 Western Blotting Mistakes (and Solutions!)

Posted in: Protein Expression and Analysis
The Top 10 Western Blotting Mistakes (and Solutions!)

As bitter experience has likely taught you, not all Western blots are pretty.  Sadly this is usually due to mistakes on the experimenter’s part. While some of these mistakes are perplexing, others are just plain dumb; but none of them have to happen to you. Read this!

1. Starting with Bad Samples

Proper protein extraction and sample preparation is critical. Bad protein samples due to improper or sub-optimal protein preparation can result in poor Western blots. While labs often have their own sample preparation methods, consider optimizing your own process. Proper sample preparation is an empirical process involving the comparison of detergent extraction versus whole cell lysate, salt concentrations, buffer types, inhibitors (to prevent degradation and loss of post-translational events), reducing agents and heating times. An optimize protocol will maximize your protein-of-interest’s solubility and stability, which will allow you to better visualize your protein and reliably interpret your Western blot data.

2. A Bad Gel

After hours of performing experiments and preparing samples, you better make sure your samples are loaded onto a pristine gel! However, even if you do everything right—you followed the recipes carefully, minded you pH, used fresh reagents, meticulously cleaned your plates—things can still go wrong with your gel. To avoid losing your samples to a bad gel, always examine your gel closely before use.

Be on the lookout for:

  • Messed up wells. Properly made wells should be uniform and rinsed.
  • Bubbles or blotting paper in the gel. These imperfections will impair protein migration.
  • A dried out gel. If your gel can be seen shrinking from the edge, it is drying out. Such a gel will not run properly.
  • A runny gel. Before you use your gel, gently tilt it to ensure that it is completely set up.

If you see any of these problems, chuck your gel and start over. It is faster to make a new gel than to do a new sample preparation AND a new gel.

3. Stupid Transfer Mistakes

While we like to think that we are above stupid mistakes, we are not. In the laboratory it is common to be multitasking, overworked and distracted—all things that lead to stupid mistakes. To minimize your number of stupid mistakes, always ask yourself the following questions before you start ANY Western blot transfer (no matter how routine):

  • Did you handle the membrane properly? That is, did you handle it with gloves, and ensure that there are no pressed marks if using laser capture?
  • Is everything in the sandwich properly hydrated? Were there no bubbles, including on the hydrophobic membrane?
  • Do you know where your proteins are? Double (or triple check) that the gel is in the correct place relative to the membrane, and that you know the orientation of your lanes.
  • Did you plug it in right? Double check that the electrodes are plugged into the correct positive/negative outlet. Oh, the lamentations I have heard when this is done improperly!
  • Check the timer. If there is a timer on the gel box make sure that it doesn’t turn your transfer off too early!
  • Watch the voltage and amps. Do not just hit “transfer” and walk away. Stay a few minutes to make sure that your voltage and amps are okay. It is never a bad idea to look for a few bubbles (a sure sign that SOMETHING is happening).

4. Disappearing Transfer

Even if you correctly set up your transfer, and double-checked the things above, a transfer can still go wrong. Transfers with swirls, mystery protein splotches, loss of protein, or a general variability in transfer efficiency are common Western blot problems. These problem are usually witnessed after you transfer when you stain your membrane and gel with Ponceau S or Coomassie for protein detection.

Luckily there ARE reasons behind these bad transfers:

  • Transferring the gel too quickly and hot. To avoid this problem, simply run your transfer at a lower voltage for a longer time.
  • Poorly fitting sandwich. A common source of transfer problems is the tightness of the sandwich. A good sandwich is tight. Make sure yours is too, by using extra sponges if needed; using appropriately thick filter paper (too thick can cause issues); and as always insure that all of your sandwich components are clean, hydrated and free from bubbles.
  • Small proteins ran away. Small proteins move faster than large proteins during transfer and can actually transfer through the membrane. If you are working with a small protein, test different transfer times and use two membranes during the transfer.
  • Large proteins never moved. Large proteins have the opposite problem. Some large proteins may not transfer at all. Consider adding a small amount of SDS to the transfer buffer and increasing the transfer time if this is your problem.

5. Bad Primary Antibody

Antibodies vary tremendously; some are fool proof while others are tricky as heck. If you are having problems getting your antibody to work, make sure that you have checked the obvious:

  • Did you get the right species? Double check that the protein you are studying really is the species you think it is. Antibody epitopes are species specific. Checking this may require some notebook and paper digging, sequencing of vectors or other types of controls.
  • Did you dilute the antibody correctly? To test this you may need to test a series of dilutions.
  • Did you use the correct buffers? Make sure you used the appropriate blocking and incubating buffer. For example makes sure you did not do anything dumb, like use the 5X concentrated buffer by mistake.

Often the best way to deal with an antibody problem is to call the company and complain. They are known to sometimes send control lysate to run, a new batch of antibody or alternate antibody for free.

6. Bad Secondary Antibody

The good secondary antibody is critical for clean and sensitive detection. It is important to use a secondary antibody that is:

  • against your primary antibody’s species,
  • has been properly stored and handled,
  • well established,
  • conjugated to the correct detection agent,
  • at the correct dilution, which in the case of secondary antibodies follows the “less is more” rule, and
  • used with the correct blocking agent.

When in doubt with your secondary antibody, borrow a well-established and loved secondary antibody from a neighboring lab.

7. Poor Development

Problems with protein detection and developing can arise regardless of the system you are using, and usually the problem lies with the quality of the developing reagents. The most common problem when using HRP conjugated secondary antibodies is that the developing reagent, which contains hydrogen peroxide, has gone bad due to age or contamination. Regardless of the type of system you are using, however, if you suspect that your reagent has gone bad, borrow some new reagent from another lab that routinely uses them and isn’t having problems.

8. Too Much or Too Little Sample

Whether you are developing by film or by laser capture you need to worry about the physical limitations of protein detection. If you have too little protein, if your primary antibody does not have high affinity or if your primary antibody is at a sub-optimal dilution, you won’t see your protein. Alternatively, if you load too much protein, your signal to noise ratio may go up causing difficulty in visualization. Additionally, if you need to quantitate your blot, you need to be even more exacting with your sample amount. In order to properly quantify, you need to make sure you are in the linear range of detection. This can be tested using serial dilutions of your sample.

9. Buffers Gone Wrong

So, sometimes you make a mistake and grab the wrong buffer or you blame someone else for making the wrong buffer: it happens. But if something goes really, really wrong and no one can figure out why, it may covertly lie with the membrane washing buffer. More than once I have seen this happen and, trust me, the figure pointing can turn brutal. But if you keep your membrane washing buffer in a clear glass container, you will always be able to see when it has gone bad, been contaminated or otherwise looks suspicious. Whenever you doubt your buffer, make your own stock of buffers, label them “Special Magic Buffer,” and see if this doesn’t get things working again.

10. You Did Not Think Ahead

Yes, that’s right, and you did not figure it out till you were all done. As laborious and redundant as it seems at times, you need to include positive and negative controls to pinpoint Western blot problems. I encourage you to always draw out your expected results (before you load your gel!) to catch any critical holes in your planning logic and allow yourself the opportunity to include all the proper controls. There are numerous types of positive and negative controls that you may choose from. The correct ones will be experiment specific, but a common positive control is a lysate expressing your protein-of-interest (usually provided by the antibody company) and a common negative control is a lysate that does not contain your protein-of-interest.  If you choose not to include positive and negative  controls, like these, know that you have lost the opportunity for a publication figure and may receive negative feedback in an upcoming lab meeting – all things to be avoided.

Now go out and make lots of pretty western blots!

Want all your Western blot buffer recipes, an ECL reagent recipe, and an easy protocol all in one place and on hand? Download our free Western blot cheat sheet.

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Image Credit: jepoirrier


  1. JackBean on April 22, 2014 at 2:21 pm

    what exactly is “membrane washing buffer”? (to which step is that refering?)

  2. JackBean on April 22, 2014 at 2:13 pm

    Still thinking what’s the “blotting paper in the gel” supposed to mean.

    • Jennifer Redig on April 24, 2014 at 3:50 am

      Referring to any contaminants in your gel. If you do not wash your glass plates well before making your gel and store your glass plates in a drawer with other junk, like blotting paper. You may find blotting paper fibers in your set gel. If you buy your gels this is not a thing.

  3. Jennifer Redig on April 9, 2014 at 9:39 am

    Thanks guys! Fixed 🙂

  4. MichaelA on April 4, 2014 at 6:38 am

    Sorry guys, but this is not a western blot, but a DNA gel. More specifically a digest of DNA with EcoRI and HindIII, which clearly do not process proteins. Or is this a bad april fools joke? Besides that, you are not giving a proper reference from where this image is taken.

  5. Greg Cary on April 1, 2014 at 5:54 pm

    Your figure is clearly not of a western blot, but represents data from a restriction digest simply due to the fact that the MW marker is in units “bp” not “kDa” or similar.

    Was this meant to illustrate a common mistake? 😉

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