phosphoSer lower on films - (Nov/08/2008 )
Hi there,
my problem is that i a looking for the total versus the phosphorylated formof a protein
both are about 100kD and the phosphoSer form appears lower on the film at about 80kD.
the total form comes out nicely at 100kD
my initial thought was that I cut the protein at the site the primary phosphoSer antibody binds but even though I reduced the time and amplitude of sonication during homogenisation it didn't change...I still get a fainter band at 100kD when I rase the priary antibody concentration to its limits
the thing is that the non-specific (if it is non-specific) folows the same increase-decrease patern between the groups as the faint band at 100kD..so could I measure it as my specific signal and use it for my results and how could I prove that this is the right thing to do?
any other suggestions are most welcomed
thanks
you will save me
my problem is that i a looking for the total versus the phosphorylated formof a protein
both are about 100kD and the phosphoSer form appears lower on the film at about 80kD.
the total form comes out nicely at 100kD
my initial thought was that I cut the protein at the site the primary phosphoSer antibody binds but even though I reduced the time and amplitude of sonication during homogenisation it didn't change...I still get a fainter band at 100kD when I rase the priary antibody concentration to its limits
the thing is that the non-specific (if it is non-specific) folows the same increase-decrease patern between the groups as the faint band at 100kD..so could I measure it as my specific signal and use it for my results and how could I prove that this is the right thing to do?
any other suggestions are most welcomed
thanks
you will save me
so, you should toy with the idea that you are analyzing two different proteins; the correlation you see may reflect kinase:substrate or phosphate:phosphosubstrate ratios
thanks a lot for your contribution so I will let you know of more info \
I have more input:
I tried to do the two membranes of the same experiment with two different antibodies one from Chemicon and one from Santa Cruz for the Ser phospho-site.
the Santa Cruz gives a beautifull band at exactly 100KDa while the Chemicon one keeps having a mind of its own.
you would say okey then keep up the good work with the Santa cruz one..the problem is that when I do the the total protein next day it is like the Santa cruz doesn't let it work and I get no signal...strange I know...although the Ser Santa Cruz is anti-goat and the total protein Chemicon is Anti-rabbit I am thinking of doing a stripping in between the two incubations and see if I can get rid of the effect...
thanks again and any more ideas would be most welcomed!!!