Hi all,
I am interested in knowing if there are established methods in:
1.) stripping the antibodies off the excised bands of a western blot (PVDF
membrane)
2.) then recovering proteins from the bands for mass spectrometry?
1st reply
I think there's a protocol which uses SDS and betamercaptoethanol
(incubate at 50 degrees, 30min) which will take off a lot of the primary
antibody. It will probably also take off a lot of your protein though.
Maybe you can
try purifying digest on immobilised protein A/G beads but I'm not sure if
that will help much.
2nd reply:
In our experience, the antibodies persist past all efforts to roeomve and
almost always preferentially ionize over the analyte of interest. It is
better to run two gels and use the antibody-visualized one to locate the
areas of interest on the 2nd. The problem here though is that the
concentration may be too low to work with by MS. A check of concentration
would be the ability to stain with Coomassie blue, Sypro Ruby, or lastly
silver. If the analyte shows up with the first two, there is enough to
work with. If it barely shows up or doesn't show up at all with silver,
there is almost no hope to see a digest from the band using MS. In our
lab, we are only having a 45% success rate with silver stained gel bands
even employing Farmer's reagent to destain. The technique we're using is
LC/MS/MS of a destained/ reduced/alkylated/ enzyme digest. MALDI is used
more for intact proteins and oligonucleotides in our lab at this time.
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