In our SILAC experiment we analyzed supernatants from cell cultures. It seems that treated cells secrete more protein than control cells (more or less 3 fold, as checked in many other cultures we have performed). The relative quantification by SILAC it's been performed mixing equal amounts of protein from both conditions. To validate this results by WB, we should load an SDS gel with equal amount of protein, however we think it is not giving us a valid info as treated cells secreted 3 times more proteins than controls. Do you think that correcting the ratios from the SILAC analysis by these 3 fold difference in protein secretion is a valid strategy? In that case I assume that for the validation by WB we should load equal amounts of volume from both conditions. Thank you in advance!
Thank you for your answer! I think, this is a valid point when performing analysis on cell lysate. However, in our case we are analysing the secretome of our cells and, as a conseqence, we cannot use a control protein as GAPDH or actin because they are not secreted proteins (actually we do not know any protein which is secreted constitutively that could be used as control).
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