A compact overview of troubleshooting guides for SDS-PAGE protein electrophoresis

One-dimensional (1D) Denaturing protein electrophoresis is a well-established and routine method in many labs dedicated to checking protein expression levels, helping to determine the size of the protein, and being essential when preparing for Western blots.

Still, getting non-smiling sharp and accurate gel images is an art.

Every researcher has an access to numerous troubleshooting guides for protein electrophoresis. flash4science has summarized the most common causes of protein SDS-PAGE electrophoresis failures and wants to share them.

1. No protein bands at all or just very faint ones

The reasons for that can be too little proteins loaded on the gel, suboptimal gel staining dye or wrong staining conditions. Load more concentrated sample.

Or maybe you need to change to silver staining instead of Coomassie Blue?

Here is our offer for gel staining - Fast SeeBand Single Step Protein Staining Solution

Do not forget to fix the proteins on the gel, follow staining protocols.

Can it be that you simply forgot to switch the gel at the right time and smaller proteins simply went out into the buffer?

2. Smiling protein gel

Nobody smiles when seeing the SDS-PAGE gel smiling. To avoid gel smiling effect you have to be patient - take care to run the electrophoresis slowly at 200 V up to one hour. But stop it when the dye front reaches the gel bottom.

3. Smeared protein bands

Maybe your protein sample was contaminated with nucleases? It happens seldom but looks terribly. So take care to work clean.

Can it be that you have forgotten to prepare the stacking gel on top of the resolving gel? Or maybe your gel percentage was not optimal.

Hopefully your protein samples contain SDS or other denaturing agents, otherwise one shall not expect good protein separation.

The most common failure causing smearing, however, is overloading of the gel wells with too much protein material. Simply reduce the sample volume or dilute proteins next time.


4. The sizes of the proteins look different from expected ones

Denaturing protein gel electrophoresis is suitable for precise protein sizing when you use unstained protein size standards. All labs like to use prestained protein ladders because they give colored bands for better orientation and allow for monitoring of protein transfer to membranes for Western blotting.

One trick connected to prestained protein ladders is that the dyes bound to proteins of the ladder change the migration of the protein. Therefore to use prestained ladders for precise protein sizing, one has to prepare the standard mobility data curve and to use it for protein size calculation. Suppliers normally provide the protein mobility data for their standards in different common electrophoresis systems such as  Tris-glycine, Tris-acetate or Bis-Tris buffers in both gradient gels and standard gels of 8 to 15%.  

Take free samples of protein electrophoresis ladders by highQu.

Protein modifications occurring naturally such as glycosylation, phosphorylation or other may also alter protein mobility, so take this into account.

Check all our partner offers for protein electrophoresis.


Here we found for you a few great troubleshooting guides and protocols for protein electrophoresis:


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Public domain images by Dawn Hudson

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