Troubleshooting SDS-PAGE Sample Preparation Issues

There are five different signs that indicate that there are problems with SDS-PAGE sample preparation: sample leaking out of the well during or after loading, samples clumping and not migrating, you aren’t seeing bands, you’re seeing too many bands, or you’re getting smeared bands.

SDS-PAGE sample preparation issues:

• Samples leaking out of the well during or after loading
• Samples clumping in the well and not migrating properly
• No bands are visible after staining the gel following electrophoresis
• Too many bands in your gel after protein electrophoresis
• Smeared bands in your gel

In this article, we will discuss the issues that commonly occur and for each of these situations, the troubleshooting steps might solve the problem.

Article table of contents:

Samples leaking out of the well during or after loading

Samples clumping in the wells and bands not migrating properly

No bands are visible after staining the gel following electrophoresis

Too many bands in your gel after protein electrophoresis

Smeared bands in your gel

General good practices for SDS-PAGE

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Protein samples leaking out of the well during or after loading

Seeing distorted and smeared bands during protein electrophoresis can be due to samples leaking out of the well, which is caused by sample overload, uneven loading, incorrect buffers, or air bubbles in the wells during loading. Proper loading helps prevent issues and leads to better results.

Figure 1. Example of how sample leakage would appear during protein electrophoresis.

First possible explanation: The loading buffer may not have enough concentration of glycerol. Glycerol in your protein sample helps them sink down inside the well during loading.

Troubleshooting suggestion: Check for glycerol concentration in your loading buffer. You might need to increase glycerol concentration.

Second possible explanation: You might need to improve your gel loading approach. Loading samples in a polyacrylamide gel might not be as easy as loading an agarose gel for DNA electrophoresis. Below are some suggestions to keep in mind.

Troubleshooting suggestion: If there are air bubbles inside the well while you load that well, the sample would spill out during loading. The way around this is to first take a little bit of running buffer and rinse the well with it before loading your actual sample. This ensures that all air bubbles escape the well before you load your sample – this is an effective way to prevent samples from spilling out of the well during or after loading.

Another common issue, especially with people new to acrylamide gels, is that they tend to overfill the wells during loading. This potentially can lead to samples spilling out.

As a general rule, you would not want to load the well more than a maximum of 3/4 of its capacity.

Also, try to load all wells with equal volume.

Protein samples clumping in the wells and bands not migrating properly

Improper band migration and sample clumping in the well during protein electrophoresis can result from loading too much protein in the wells, high salt or detergent concentration, or protein aggregation or precipitation in the wells.

Figure 2. Illustration of what improper migration would look like.

First possible explanation: Too much protein was loaded in the wells.

Troubleshooting suggestion: Check the concentration of proteins in your samples. A good practice is to load 10 µg of protein per well.

If too much protein is loaded in the wells, band resolution will also usually be poor.

Second possible explanation: Protein aggregation or precipitation in the wells.

Troubleshooting suggestion: Ensure the solubility of your proteins in sample lysate before loading them into the gel. In this case, take extra care during the extraction of your sample proteins by doing proper homogenization.

One way of doing this is by sonicating your sample source (cell culture, bacterial culture etc.) adequately followed by centrifugation to remove cell debris.

Another suggestion is to add DTT or BME in your lysis solution. These chemicals reduce protein aggregation by breaking secondary structures.

Heating your lysate also aids in getting your sample proteins into their primary structure and reduces aggregation.

If your sample proteins are hydrophobic, there are higher chances of their aggregation. For this, consider adding 4-8M urea in your lysate solution before loading.