WB: one single band but at the wrong size... - what to make of it? (Jul/22/2005 )
Hi all,
I'm doing WBs to detect RAGE (a membrane-bound receptor) using a monoclonal ab. I'm getting one single sharp band, which is good... My problem is that the size is completely wrong (approx. 60kD difference).
I also did IHC on cultured cells with the same ab and get nice stainings that look absolutely comparable to stainings shown in the literature or by manufacturers.
Any ideas of what to do with it or how to continue?
Thanks a lot in advance,
Günther
is the band ~60kDa smaller or larger then expected?
How are you preparing your protein for WB?
I think we need more information about your experiment to troubleshoot.
1. Before Western blot, you did a native gel or a SDS-PAGE? (I guess it's SDS-PAGE), in SDS-PAGE, sometimes 10kDa difference is still acceptable.
2. Have you checked up your protein of insterest carefully? any subunit?
3. Have you included 100mM beta-mercaptoethanol when boiling samples?
4. A clear band on Western blot doesn't mean it is the protein of interest. My own experience tells me that false postive could appear, even much stronger than the real one! Furthermore, if you are using inappropriate extraction buffer, the desired protein won't show up.
1. Before Western blot, you did a native gel or a SDS-PAGE? (I guess it's SDS-PAGE), in SDS-PAGE, sometimes 10kDa difference is still acceptable.
2. Have you checked up your protein of insterest carefully? any subunit?
3. Have you included 100mM beta-mercaptoethanol when boiling samples?
4. A clear band on Western blot doesn't mean it is the protein of interest. My own experience tells me that false postive could appear, even much stronger than the real one! Furthermore, if you are using inappropriate extraction buffer, the desired protein won't show up.
Thanks for your replies!
ok, here come the additional infos:
- the band appears at approx. 120 kD instead of approx. 50 kD
- yes, the gel is a SDS-PAGE (12% running gel)
- studied the literature, some claim a double-band around 50 kD, most find a single band around 50 kD
- yes, b-ME has been added prior to boiling the samples
As for the protocol: the cell were detached with trypsin/EDTA, washed in PBS and lysed in the loading buffer (Tris, b-ME, SDS, glycerol). After blotting the membrane (nitrocellulose) was checked with Ponceau S and everything looked fine. Incubation with a mouse-mab was followed by ECL detection.
As said in my first post, IHC with this ab looked as others found in comparable systems.
Thanks a lot or your replies and I hope I provided all the infos that you asked for.
Günther
sorry for the shameless bump... 