3 replies to this topic

[mac1]

I would like to know if a gel after being stained by coomassie blue, and then destained acoordingly, could be used for transfer onto the membrane. Reason - Would like to know exactly how much protein was eluted from the gel and then onto the membrane. This was done by comparing the coomassie blue stain with the ponceau stain pattern.

I welcome feedback and comments.

[Simonsays]

The thing is that coomassie and destain contains MeOH and acetic acid which fix the proteins, precipitate them in the gel. The transfer to a membrane becomes then very difficult. You should first transfer and the color the remaining proteins on the gel with Coomassie.

Simon

[mabusheh]

Simonsays, on Mar 16 2005, 02:02 PM, said:

The thing is that coomassie and destain contains MeOH and acetic acid which fix the proteins, precipitate them in the gel. The transfer to a membrane becomes then very difficult. You should first transfer and the color the remaining proteins on the gel with Coomassie.

Simon

ohh, does the same hold true for gels stained with Sypro.

[badcell]

SYPRO Tangerine, but not SYPRO Red or SYPRO Orange can be used to stain proteins in the gel before blotting. According to Molecular Probes:

Quote

SYPRO Tangerine protein gel stain provides fast, easy and sensitive detection of proteins in gels (down to 4 ng/band) without the need for fixatives. Staining is compatible with subsequent Western blotting, zymography, electroelution or mass spectrometry. Compared to Coomassie Brilliant Blue and silver staining, SYPRO Tangerine stain provides more consistent protein-to-protein staining and a much broader linear quantitation range (over three orders of magnitude). Stained proteins can be viewed with a standard UV or blue-light transilluminator or with a laser scanner. For optimal sensitivity with Polaroid film, use of the SYPRO protein gel stain photographic filter (S6656) is recommended.

Cheers!