Carrier protein in target protein - (Apr/12/2007 )
Anyone have/had any problems with running westerns or SDS page gels with recombinant proteins contianing a carrier protein? My recombinant mouse Oncostatin M contains BSA as a carrier protein which stabalize the protein which is nice but I also get this fat band around 67kd when my OSM band should be around 21kd when I run it on a gel. When i do a western, I get the same fat band and the 21kd band is missing. Anyone experience problems with carrier proteins or am I doing something wrong? :-/
-Rubyeye
-Rubyeye
Hello Rubyeye!
as I know carrier - is not additive but it is a molecule which conjugated with your protein. Is it so?
If it is not so and BSA only as an additive protein I can't understand why do you see protein BSA on western because of your Ab for OSM only?
as I know carrier - is not additive but it is a molecule which conjugated with your protein. Is it so?
If it is not so and BSA only as an additive protein I can't understand why do you see protein BSA on western because of your Ab for OSM only?
Yea I'm starting to wonder if OSM is getting conjugated with the BSA. Is there any way to dissocatate the proteins? Wouldn't the sample buffer with mercapotethanol and SDS dissociate any protein-protein interactions? Maybe I just need to buy the carrier free OSM with half the stability.
-rubyeye
as I know carrier - is not additive but it is a molecule which conjugated with your protein. Is it so?
If it is not so and BSA only as an additive protein I can't understand why do you see protein BSA on western because of your Ab for OSM only?
Yea I'm starting to wonder if OSM is getting conjugated with the BSA. Is there any way to dissocatate the proteins? Wouldn't the sample buffer with mercapotethanol and SDS dissociate any protein-protein interactions? Maybe I just need to buy the carrier free OSM with half the stability.
-rubyeye
Conjugated proteins bind each other covalently. In some cases bond may be acid-labile and destroy with time ( concern hydrazide and Schiff base linkage if they are not reduced specially)
I think it is not possible to break this link with BME. But if it is conjugate so you could'nt see band like 67 according BSA free and band according 21kDA - your protein ( you can see them in a case when conjugate was not purified after excess of conjugated proteins or conjugate degrade a little bit) but you should see band near 90-100kDA region or on the start if conjugate consist of 2-3 molecules of your protein with BSA. If you don't see this bands ( according your conjugate) on PAAGe so it is not conjugated protein. Try to remake PAAG with and without BME in samples ( better gradient gel 5-20%)
your protein may be extremely low in concentration compared to the carrier (most likely bsa, used to stabilize the protein) and you may have to load much more on the gel. this will make the large band really huge and may disrupt stuff on the sides.
there is also the possibility that the lot of protein that you purchased is no good. call the company and ask them to check the lot.
If you buy cytokines like oncostatin M from R&D you can normally buy a carrier-free preparation. This might help you with your Western. One other thing you could try is to use 5%BSA/PBS-Tween-20 as a blocking buffer instead of using powdered milk this should reduce your non-specific BSA staining and may allow you to get cleaner results for the OSM band.
All the best,
Ceri