Loading of sample to SDS-PAGE - (Feb/08/2005 )

hello everybody,
I can not decide what is the "best" way to measure the amount of protein prior to loading on SDS-PAGE....Should I use only assay (e.g. BCA assay) or is it enough to check bands after Ponecau staining and to decide by eye? Or should I combine both? My advisor does not care which one from those 2 methods I will use...but.....
thanks in advance
Martin

hi
honnestly only using ponceau staining is not a good alternative because the human eye is not precise as a BCA assay (i mean bradford). and if you want to reproduce your experiments, ponceau stainig is not very useful.
And doing a BCA assay before loading can preserve time : for example if you r ponceau staining reveals not equal amount of proteins you must restart your sds page...
You cant do both if you want but i think that a pre loading amount measure is essential.

hope have been useful for you.

i personally prefer to do both:

before the PAGE i check protein content by a commercial assay, to get an idea how much protein is there. after transfer i do ponceau staining to check if the protein assay has worked well and the transformation efficiency was ok, since sometimes the outer lanes seem to transfer not as well as the others....

BTW: I asked before around here, and got no answer.
does any one know the exact mechanism of ponceau stain? I.e. is it a chemical or physical stain and what aminoacids interact with ponceau, etc.?

mike