Protein loading control for RAW cell supernatants - Western blot loading control (Nov/03/2006 )

Hi,

I'm using supernatants from RAW cell cultures (untreated and treated with LPS) for my western blot. Can anybody suggest a suitable protein loading control (such as beta actin or GAPDH) for RAW cell supernatants?

If I use lysates, I think I can use beta actin, but will it work for supernatants?

I have been using Ponceau S staining to ensure equal loading, but I don't know how accurate it is, although I see nice bands (and some differences in intensities - I loaded 8 samples - four control and four treatments - all the four controls had equal loading, and all four treatments had equal loading, but the intensities of the control and treatment samples were different, adding more to the confusion ) any suggestion in this regard is greatly appreciated.

RR

I think it would have to be a secreted protein (i.e. actin, GAPDH etc. will not be suitable). I'm not sure of one that won't change depending on the stimulus you give. There are cell viability/number assay based on measuring secreted proteins e.g alamar blue. What are you stimulating the RAW264 with? Presumably an inflammatory stimulus which will increase secretion of proteins (e.g cytokines) into the supernatant which is probably why you have different amounts of protein.

If you're loading equal volume of supernatant then you results should be fine as long as you have the same number of cells per stimulation. Maybe you should make a protein lysate of the cells and confirm that there is a equal amount of protein in each cell lysate by Bradford/BCA assay.

Ceri