Dear all,
I try to prove the presence of the LDL - receptor.
I performed my Western Blot several times, with different antibodies and I always received the same bands.
One of these bands (between 25 and 35 kDa) is very strong and after quite a long research, I found out, that this band is most likely a splice variant of the LDL - receptor.
I would like to verify this assumption by a MALDI - TOF MS analysis. My problem is, that I have no idea, how to extract this specific band from my gele after SDS - PAGE, as there will be many other bands, which will overlap my band of interest. Or is there any fast and easy way to extract the band after incubating the membrane with the AB´s ?
Best regards,
Bastian
Bastian, what does the gel look like when you stain it with silver and/or Coomassie? If you see the same discrete band there, it is worthwhile to perform an in-gel digest with trypsin and analyze with MALDI-TOF using Peptide Mass Fingerprinting and (instrumentation allowing) confirm some of the assigned peptides by MS-MS or at least PSD.
If the band pattern from a global staining looks completely different then your protein of interest is very probably a minority compound and as such not amenable to PMF or MALDI-TOF. Try and find somebody who runs high-quality nanoLC-MS/MS instead which can resolve up to 150 different proteins in a gel band in many cases. Am not sure who would be able to do this in Bielefeld - the Niehaus group might be able to help.
Dear Christof,
thanks for your message.
To be honest, I never stained the gel after performing the SDS - PAGE. But I will do it the next time. So, what if I don´t see the band of interest in such a discrete way as in my Blot?
Could I give it a try to cut out my band and analyse it via MALDI - TOF after digesting it with Trypsin? Through the Trypsin, I should receive a Peptide Mass Fingerprinting, shouldn´t I?
Best regards
Bastian
Bastian, is your antibody good for immunoprecipitation? If so, just IP from a large amount of cells, run 95% of the IP on a gel, stain the gel with something like Coomassie (or silver if you don't have too much material) and then cut out the band and give it for MS. I'd first run a western on the other 5% of the IP just to confirm the band is reactive with your antibody. There will still be other proteins in the region, but your protein should be a majority component during the MS. Remember not to include DTT when eluting after the IP, as you don't want the light chain of IgG which will run around the same size as your protein of interest.
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