Zones of white around protein bands on SDS-PAGE Gels - has anyone seen this before?

Hi, your assumption about the salt may be the cause.

I don't know if my experience can help you, because I didn't use tricine gel or imidazole. I use coomassie in methanol and acid acetic and destain methanol and acid acetic because it was better than water.

In my case after the destain I have and uneven coloration, white spot random on the gel, I can't understand what it is the problem. I read that the SDS in the gel can interfere with the coloration and I had to wash well the gel in water before the coloration in Coomassie.

From your image I saw that in the wells 6 and 7 (samples without imidazole or less salt) you didn't have the problems. I don't know if you can prepare the peptide without imidazole but at least try to wash the gel before the coomassie stain. it is only a possibility but you can try.

I hope it works out for you.

Valeria

Leisha McGrath

Hi Valeria,

We have tried to stain in methanol/acetic acid Coomassie before, but the methanol causes the protein to disappear from the gel; apparently using methanol in Coomassie makes smaller proteins dissipate from gels.

The gels are rinsed with distilled water after electrophoresis to get rid of residual SDS from the running buffer, and then they are rinsed with distilled water after fixing. We are wondering if it is a salt issue now; I am running samples of the buffers used, without any protein in them, on a gel to see if the same issue occurs again when staining. Then we may be able to determine if it is a salt issue.

Unfortunately, my protein doesn't seem to be stable when imidazole is absent; right now, we are trying to find out the lowest concentration of imidazole we can use that will keep my protein stable.

Thanks so much for your advice!

Leisha

Nikolay Klyashtornyy

The presence of white zones or halos around protein bands on SDS-PAGE gels can be caused by various factors. While it's difficult to pinpoint the exact cause without further investigation, here are a few potential explanations:

Protein aggregation: The white zones could be due to protein aggregation. Aggregated proteins may appear as diffuse white areas surrounding the protein bands. Aggregation can occur during gel electrophoresis or during the staining process. Factors such as high protein concentration, improper sample preparation, or excessive handling of the gel can contribute to protein aggregation.

Staining artifacts: Staining artifacts can also lead to the appearance of white zones. These artifacts may arise from interactions between the staining dye (e.g., Coomassie G250) and other components in the gel or buffer system. It's possible that the imidazole or high salt concentration is interacting with the staining dye, causing the formation of white zones.

Incomplete destaining: Inadequate destaining of the gel can result in the presence of white zones. If the gel is not thoroughly destained, residual Coomassie dye can accumulate around the protein bands, creating a white appearance.

To troubleshoot and narrow down the cause of the white zones, you could try the following:

Adjust staining conditions: Experiment with different staining conditions, such as altering the staining time, concentration of Coomassie dye, or pH of the staining solution. This may help identify if the staining process is contributing to the white zones.

Improve destaining: Ensure that the gel is adequately destained using fresh distilled water. Extend the destaining time if necessary to remove any excess dye.

Modify sample preparation: Optimize the protein concentration and sample preparation protocol to minimize protein aggregation. Consider using a denaturing agent, such as urea or SDS, to ensure proper solubilization of the protein.

Verify gel quality: Check the quality of the gel itself, including the gel composition, polymerization process, and storage conditions. A well-prepared gel can help minimize staining artifacts.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

Leisha McGrath

Nikolay Klyashtornyy many thanks for your answer, it is very detailed which is much appreciated!

I have tried longer destaining, and this does seem to help in reducing the effect. I will keep an eye on things, as we use Coomassie G250 and some others have noticed the halo effect in samples loaded to gels where there is no imidazole present.

Thanks you again!

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