I'm wondering if the methanol and acetic acid impedes the elution of proteins in an electrphoresis gel. I know methanol fix proteins, but do this is a permanent fixing? and what about the coomasie molecules, do someone know how strong are the interactions with proteins?
I don't think Coomassie staining is a problem to elute a protein. I already sent for identification proteins removed from a Coomassie-stained electrophoresis gel SDS PAGE and the results were good!
I didn't extract the protein, but the lab that analysed my proteins asked specifically for a band from a Coomassie-stained electrophoresis gel SDS PAGE.
Yes, it is possible to elute a protein from a Coomassie stained electrophoresis gel PAGE SDS. However, the methanol and acetic acid used in the staining and destaining process can affect the ability to elute the protein from the gel.
Methanol is used in the initial fixation step to denature and precipitate the proteins, making them more visible during staining. It can also crosslink proteins, making them more difficult to elute from the gel. Acetic acid is used in the destaining step to remove excess Coomassie dye, but it can also acidify the gel, which can cause the proteins to become more insoluble and difficult to elute.
To overcome these issues, different elution buffers can be used to maximize the recovery of the protein of interest. For example, Tris buffer with 0.1% SDS, or acetic acid with 50% methanol can be used for elution.
Regarding the interaction between Coomassie dye and proteins, the dye molecules bind to basic and aromatic amino acid residues in the protein, resulting in a blue-colored complex. The binding is relatively strong, but can be disrupted by high concentrations of salt or detergents, allowing the protein to be eluted from the gel.
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
thank you Joana B. Caldeira and Nikolay Klyashtornyy for your rsponses. I'll try the elution buffer Nikolay Klyashtornyy mentioned.
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