Unable to identify our protein - need an idea (Apr/19/2007 )
Using western blot, we have identified in serum a neuronal secreted protein. We have seen the same band with polyclonal and monoclonal Ab. To be 100% sure that what we are studying is that protein, we want to identify it by mass spectrometry. This is not absolutely essential, but it would be a very good complementary experiment.
However, that´s being difficult. We have depleted albumin from serum and then the IP the protein, run a SDSPAGE gel, stained it with coomasie, saw 4 bands, including the expected one at the MW of our protein, and sent it to the mass spec service to confirm it.
But it couldn´t be identified because there was too much antibody, even using an IP kit which it´s said that allows you to obtain your antigen without Ab contamination 
Another possibility would be to run a 2D gel, but how to be sure of the spot even knowing the pI and the mW? serum is a very complex mixture of proteins.
So now we don´t know how to identify it. Any ideas? 
thank you
However, that´s being difficult. We have depleted albumin from serum and then the IP the protein, run a SDSPAGE gel, stained it with coomasie, saw 4 bands, including the expected one at the MW of our protein, and sent it to the mass spec service to confirm it.
But it couldn´t be identified because there was too much antibody, even using an IP kit which it´s said that allows you to obtain your antigen without Ab contamination
Another possibility would be to run a 2D gel, but how to be sure of the spot even knowing the pI and the mW? serum is a very complex mixture of proteins.
So now we don´t know how to identify it. Any ideas?
thank you
Did I get it right, your bands are at 25 and 50 kd? Because otherwise the AB shouldn´t disturbe the ms...
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Did I get it right, your bands are at 25 and 50 kd? Because otherwise the AB shouldn´t disturbe the ms...
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Yes, the band I want is at 50 KDa
1. Ask the ms-people for MRM: With the sequence of your protein only these peptide-signals will be analiesed via MS/MS. This methods works with low abundant proteins in the presence of high abundant proteins.
2. Dyna-Beads for "fishing" your protein and elute the pure protein without AB!!!
thanks ms-olli. I will ask them.
what they had told me is that they tried to digest some peptides which hadn´t matched with IgGs but they said that no reliable results were found 
dyna-beads seem a good idea, I´ll tell it to my supervisor. thanks 
How much purification have you done? If you can get a clean band by SDS-PAGE, all you have to do is cut out the band and send that off for analysis.
yes, that´s what I did. I saw the band at the MW of my protein staining with coomasie, I cut and send it and they told me that there were too much IgG and the couldn´t identify another protein different from IgG.
If your band is a mix of IgG and your protein of interest, you might be able to take the IP fraction, dissociate the complex and purify by HPLC (ion exchange, reverse-phase might work best), then run the fractions on a gel and send off your multiple-purified protein
thanks swanny, that seems a good option
I haven't ever tried anything like this, but if you run 2 2D gels and transfer one to a Western membrane and identify your protein of interest with your monoclonal/polclonal ab. Then identify this protein on the other gel which is coomasie or sliver stained and cut it out for MS. Would that work?
Ceri