Hi,

So there are several reasons to not see any protein bands on the gel.

1. First , You might have diluted the protein sample too much hence you cannot observe it on the gel. Also check if the quantification is proper. Next time when you load the same sample do serial dilutions and then load each dilution. This can give you an idea of how much concentration should be loaded. Generally 10-20ug protein concentration can be observed on the gel.

2. Second reason can be, the medium in which the protein was diluted. The water might have had proteases which could have degraded the protein. Try using protease inhibitor, this will prevent the protein from getting degraded.

Hello Verônica Assalin Zorgetto-Pinheiro

This could be a sensitivity issue to your protein of interest. Maybe the protein concentration is inadequate for coomassie detection. Increase the sample concentration and check. If that is the reason, staining with colloidal coomassie could simply solve your issue too. Furthermore, load an adequate amount of protein into wells. Check for any protease contamination, and make sure samples did not freeze-thaw. If these changes didn't work, I see silver staining as a good idea.

The following attachment also provides in-detail troubleshooting for SDS-PAGE. Hope this helps.

https://docplayer.net/5656374-Gel-electrophoresis-of-proteins.html

Best wishes!

Hard to say without providing details of the protein, I can see protein with a size equivalent to the lower molecular weight protein of the ladder.

Omar Gonzalez-Ortega That continuous band aligning with the lowest molecular weight band of the marker is probably the dye front.

Verônica Assalin Zorgetto-Pinheiro Are you dealing with an unknown protein or do you have an expected molecular weight?

Staining with bromophenol blue (coomassie)...

This is not correct, two dyes are not the same thing. It seems you used a colored marker and proteins in the marker cocktail are already dye-attached. (e.g Bio-rad Precision Plus Protein Dual Color Standards)...I would agree with others' indications if you did not use bromophenol as a visualization stain before imaging because it won't interact with proteins.

Thaks a lot for your answers. I’m working with a fruit’s protein concentrate and the samples are steps from the enzymatic digestion of it.

I’ll discuss the next steps with my colleague.

Once again,

Thaks!

You are mistaken Bromophenol blue is not Coomassie Brilliant Blue.

This are two different dyes which serves different purposes:

Bromophenol blue does not interact with the DNA or protein molecules being analyzed. It simply aids in visualizing and tracking the progress of the electrophoresis run.

Bromophenol blue is a commonly used tracking dye in gel electrophoresis. It serves several purposes in the electrophoresis process:

Coomassie dye, specifically Coomassie Brilliant Blue, is commonly used in gel electrophoresis for protein staining. It serves the following purposes:

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