MUC1 staining (DAB) has been successful for IHC, both with tris and citrate buffers, however, western blot have failed miserably. CST
VU4H5 antibody is compatible for both IHC and WB. Is stability an issue for this protein in cell lines ? I am using VU4H5 (1:500) and secondary ab, goat anti-mouse IgG-HRP sc-2005 (1:2000).
Sadly Samu
in my experience just because an antibody works in IHC doesn’t mean it will work in Western blotting even if the data sheet says otherwise. I would suggest 2 things
- unfortunately when Western blots do not work there isn’t much you can do all things being equal other than try another antibody
- I say all things being equal because the following might help
Sadly Samu
in my experience just because an antibody works in IHC doesn’t mean it will work in Western blotting even if the data sheet says otherwise. I would suggest 2 things
- unfortunately when Western blots do not work there isn’t much you can do all things being equal other than try another antibody
- I say all things being equal because the following might help
Keep in mind that when running the Western blot, in most cases you are trying to detect a protein that has been denatured and linearized (assuming of course, you've run SDS-PAGE). The tertiary and/or quaternary structure of the protein may not be intact enough for the antibody to recognize it's epitope anymore.
In IHC, you are essentially detecting the native protein, with all folding and 3 dimensional structure intact. My suggestion would be try another antibody.
That is a good point you raise Joseph and sometimes - in fact most of the time in my experience - another antibody is usually the only effective strategy
Mucin proteins are huge ~300kDA and often dimerise to form #600kDA molecules. what size are you looking for? what gel % did you use? What did you use as a lysis buffer?
Thats useful information Michael
- Under standard SDS PAGE conditions with say a 6-20% gel you struggle to resolve and transfer proteins much above 150kD in my experience
- If you are attempting to detect proteins that are 300-600kD you would need a very low % acrylamide gel
- In addition, you might want to perform wet transfer for at least 3 hours or maybe even on ice or in the cold room overnight !!
When I used to run gels on human MUC1, we would run 10-20% gradients and basically use the 125 kD marker as the dye front (ie: run the 125 kD standard band to the bottom of the gel). There are also some protocols for agarose vertical mucin gels... but I never had much luck with those.
As Michael noted, mucins can be HUGE - and they also exhibit variable glycosylation of tandem repeats. So keep in mind that when/if you get a signal, it often times isn't a single band, it's a smear, since the glycosylation can be so different.
Laurence mentioned one very important and often overlooked Western step - TRANSFER. Make sure you have enough protein in the gel, and then work out the transfer conditions (voltage and time) considering the size of the protein. Also, make sure your transfer is assembled correctly, - do you see the protein ladder on the membrane?
Three tips for the transfer optimization:
- use a high molecular weight protein ladder,
- two membranes, and
- start with the overnight transfer (16 h) under standard conditions applicable for your device. Good luck.
The horror of white wb! I had a terrible experience with other protein...i felt so desesperated! At the end was that the antibody was so arggg and i change blocking membrane with milk and using milk to prepare the antibody to block and prepare the ab solution with BSA and it Worked.
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