Could anyone give me some advice about the elution of protein from acrylamide gel. I have searched some protocol, but I am still confused about the elution buffer they used in those experiments, some used SDS-PAGE sample solution, some did not give detail component of the elution buffer. I am looking for a technique that enables to elute protein with high yield.
Thank you so much for all your help and suggestion!
This instruction manual for the Bio-Rad electroeluter may be helpful regarding elution buffers.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652976B.pdf
You could prepare a cleavable electrophoretic gel. Instead of bis-acrylamide, use one of the cleavable cross-linkers such as bis-acrylylcystamine or dihydroxyethylene-bis-acrylamide.
This instruction manual for the Bio-Rad electroeluter may be helpful regarding elution buffers.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652976B.pdf
I used in the past this passive elution protocol from thermofisher (https://tools.thermofisher.com/content/sfs/brochures/TR0051-Elute-from-polyacrylamide.pdf). The protocol worked well for my need (Identification of band identity by ELISA), but i dont know about the yields of the method (Probably low). You can try this, its very easy...
Thank you so much for all your help.
Mr. Ramon Roman Roldan, now I am also trying with the procedure described in the file you support. But the further use of eluted protein in my experiment is quite different, I want to run the eluted protein again in SDS-PAGE for further identification. The technique i am going to perform for this further application, however, is not much sensitive, this is reason why I need to use a high concentration of protein.
Today, I started the experiment, but one trouble I got was that the gel was not totally immersed in elution buffer, I guess the reason is because of the high volume of gel.
So I wonder whether there is some calculation to ensure the sufficient volume of elution buffer for the equivalent volume of gel? Could you please give me some advice based on your experience?
Again, thank you all so much for your suggestions.
Best regards,
Hoang Thi Xoan
Well, normally, i don't do any calculation, i only put the band in the tube, add a small quantity of buffer (50-100microL) and then i crush the slice with the plunger of a sterile tuberculin 1ml syringe (But taking out the black rubber of the end). I think that is the most important part for achieve the maximun surface submerged in the buffer and thus improve the yield. Once the slice is crushed you can add the buffer necessary for totally submegerd it, but if you add too much buffer, you probably dilute the protein too much and thus you probably will have to concentrate your sample with an amicon tube of similar.
Following this protocol, normallly i can view the protein in a gel, but i stain the gels with Oriole Fluorescent stain (Biorad) wich is quite more sensitive than coomasie, so if you dilute too much your sample, you will have to go to more sensitive stains like silver o fluorescent.
Best regards
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