HELP: Excision of gel band from EMSA gel - (Dec/28/2006 )
I am purifying a DNA-binding protein from bacteria.
Right now I have my protein concentrated several hundred times after a few chromatography steps.
I would like to know, up till this step (elimination most contaminating protein), can I simply run an EMSA gel, and then excise the gel band, put the gel slices into a SDS-PAGE gel and run it?
I want the sample for MS/MS
I have tried other chromatography steps, but they are not successful, and my time for graduation is limited!!!!!
I have considered elution the protein in the EMSA gel slice, but will this further cost some lost of my sample? Do you think directly put the gel slices into a well of SDS-PAGE will work? Anyone tried?
it can work but:
you have remember to equilibrate the gel slice with sample buffer;
you have to push the gel slice all the way to the bottom of the well;
you have to realize that the protein will exit the slice more slowly than it will pass through the stack which may lead to smeared bands (if the slice is large).
you can homogenize the gel slice in sample buffer and load the homogenate into the well but be aware that this homogenate is difficult to handle.
or you can homogenize the gel slice in sample buffer (and heat if necessary) then centrifuge and apply the supernate to the gel.
we have done all of these and have had success with all. but the last procedure that i mentioned may have been best.