I am currently performing western blots on my protein of interest, which is about 300kDa in size. The protein is secreted from my cell line and so I collect the cell media, concentrate my protein by spinning down the media in filter centrifuge tubes, and then perform the western on the concentrated material.
I have been able to detect my protein this way, however now I am attempting to suppress production of the protein. In order to show this suppression (ie the difference between samples treated with targeting and non-targeting constructs) I need a loading control, but I am having difficulty finding something suitable. Preferably I need a high molecular weight protein which is secreted from my cell line, but I have not been able to find something suitable in the literature.
I was thinking of adding a certain amount of a known protein to each of my concentrated media samples (ie targeting and non-targeting) and then probing for this protein with an antibody during the western. This should tell me if I get equal loading in all wells of my western gel.
If the media I have used was also taken from cells which were 100% confluent in all cases, then should all this be enough of a control to prove that differences between my targeting and non-targeting developed bands are due to suppression?
Also does anyone have any suggestions of what protein standard I would be able to add to my samples and probe for? BSA would be ideal as I have some available here in my lab, but I think it is too small as it would probably be run off my gel, and so I need something larger.
I'd really appreciate opinions/advice.