can we purify the protein from westernblot membrane? - (Oct/02/2006 )

Hi,

I am just thinking if there is a method that can help use to purify the proteins from the western blot PVDF membrane. Just like purifying the pcr product from agroase gel, cut it off then purify it.

Thanks.

what you can try is electroporation; as I suppose that you have not such an apparatus you have to extemporize; I suggest to use a small slide-a-lyzer with appropriate cut-off, take a DNA electrophoresis apparatus filled with your Wb transfer buffer and run the system at high ampere and maximum voltage for several hours; cool your system! larger proteins may break;

another possibility is to trypsinize your PVDF-bound protein but you will only get tryptic peptides; for Ms/Ms it will do

i was also wondering about "electroporation??" ....electroelution, i think it should work better then cutting and eluting (which gives poor yield of protein)

but are you cutting out of SDS gel? your protein will be denatured. you have to do native gel to get non-denatured protein.

i was also wondering about "electroporation??" ....electroelution, i think it should work better then cutting and eluting (which gives poor yield of protein)

but are you cutting out of SDS gel? your protein will be denatured. you have to do native gel to get non-denatured protein.

hello,

you can elute your blotted protein using DMSO. i'm guessing you want to remove this band for mass spec. if that's the case, be sure this is a pure protein. if it's a protein that's been run out on a 1D gel as a part of a complex mixture, you're headed for a mess unless it's very highly abundant.

typically, protein yields are low from PVDF/nitrocellulose extraction. your better bet would be to run 2 identical 2D gels of your extract, blot one, probe it w/ your Ab, and then stain the membrane w/ coomassie. you would then overlay your film (or, if you're using colorimetric detection, mark your spot w/ a pin prick) onto the blot to locate your spot of interest. you could then compare the staining pattern on the 2D PVDF to that of the corresponding 2D gel and cut the spot out and perform in-gel digestion. this will give you a higher amount of starting protein to digest (if you go too low, you're only going to see autocatalytic trypsin peaks in your MS data), and a cleaner preparation, which would minimize contaminating peptides that would interfere w/ MS analysis.