I am separating a family of proteins, containing many isoforms with minor molecular weight differences (MW range between 125 and 135kDa) using the phos tag technology and then following with traditional western blot analysis. I do not have an antibody that recognizes each individual isoform I am currently using a pan antibody (picking up all isoforms) and phosphospecific antibodies to the identified phosphorylation targets.
When I separate via phos tag SDS PAGE and probe with my antibodies, I obtain several bands. So phos tag SDS PAGE separates via phosphorylation state but with molecular weights so close how can I be sure the band I am looking at is referring to a different phosphorlation state of the same isoform or simply another isoform? How does molecular weight separation factor in to the equation?
fiendishly difficult. How much material do you have? if enough to visualise on stained gel - can you pull down the protein x and then the phosphorylated forms, resolve them on gels - isoelectric gel focussing may give better resolution - cut out bands, digest with phosphotase (immobilised? In gel digestion? ) resolve on gel (cut bands and do mass spec?) -this might tell you if the band you started with is from a single isoform or from 2 isoforms with different phosphorylation same mol wt.
I might be talking out of my hat here as this is way out of my area and I haven't had a coffee yet ...(apologies if I am..)
good luck
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