Dear Researcher
Please, If that possible to extract and purify a protein from SDS page Gel staining with coomassie Bleu.
Many thanks
No, you won't be able to extract proteins from a coomassie-stained gel. Part of the staining process is fixation of the protein to the gel itself. You would need to use an alternative stain like InstantBlue or SyproRuby, which don't cause protein-gel crosslinking.
1/12/17
Dear Larbi,
We did something like this a few years ago. We ran SDS-PAGE gels on a partially-purified bacterial extract. We stained the gel w/ Coomassie Blue and identified the protein band we wanted to evaluate. The stained band was carefully excised from the gel w/ a clean razor blade and put into a clean microcentrifuge tube. It was then macerated in a buffer to release the protein, which was then exposed to trypsin which cleaved the protein into oligopeptide fragments that were analyzed by LC/MS. The fragmentation pattern was evaluated using a computer program that matched the fragments to a known protein. In so doing, we were able to confirm the identity of the protein we were studying.
There is something else that you can try. See the attached reference. This describes a different Coomassie Blue staining recipe that stains proteins in PAGE gels within a few minutes without staining the gel itself. After immersing the gel in the stain, you can see bands within ~10 min. The background (gel) remains clear and there is no need to destain. In your case, you can run your protein in several adjacent lanes on a slab gel, after which you recover the gel and cut out one lane containing your protein. This will serve as a "sacrificial marker". Set the rest of the gel aside. Immerse the lane in the CBB. You should see a band corresponding to your protein in a few minutes. Recover the marker and rinse off the stain. Then, place the stained lane alongside the remaining (unstained) portion of the gel. The stained band will tell you where your protein is in the unstained portion of the gel. You can then excise this part of the gel w/ a razor and extract the protein without exposing it to any stain. Keep in mind that if you do SDS-PAGE, your protein, although pure, will be denatured by the SDS and will probably not have any biological activity.
I hope this information helps you.
Bill Colonna, Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA [email protected]
Hello Larbi. It is also possible to electo elute proteins from CBB-stained gels. Similar to the approach described by Dr. Colonna, one stains the gel, excises the region of the gel containing the protein of interest, and places this into the electroeluter. The problem with this approach is the need for an electrocution system. These are are commercially available. This will not work if you have actually used a cross-linking agent, such as glutaraldehyde, to fix your gels.
Hi Larbi,
I agree with Dr. Colonna, you can elute it using the 1st method I have used that techique, my only additional comment would be to be extremely careful with gel preparation (if you make your own) and the buffers . I would recommend using fresh gels and buffers to avoid contamination with Keratin in your samples.
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