Dear all,
We tagged a chemokine receptor with GFP and expressed this in an epithelial cell line to analyze receptor trafficking. This works good, the receptor is expressed, we do see receptor internalization and ATP changes after challenging. We also used this receptor for co-IP of a new ligand. Also the co-IP works fine as we could detect the new ligand after fishing using anti-GFP beads. However, we do have a problem with the molecular size of the receptor. The receptor itself has molecular size of 43 kD, with GFP we expect a size of 69 kD, however, what we see is a size of approximately 100 kD, sometimes even higher. Why? Any explanation? We checked the plasmid for orientation and that it contains only one GFP. Thus, I do not believe that we have a construct with two GFP which would also add up to 100 (receptor: 42 + GFP: 28,5 x 2 = 99). Any other explanations?
Hi, chemokine receptors undergo several modifications like glycosylation, palmitoylation, ubiquitination, and sulfation. So, depending on the type of receptor, the modifications affect the final protein mol.wt.
Did you see the mol.wt difference in Coomassie staining? or western?
Does the receptor alone, without GFP, show the expected size in the Western blot?
Dear Loka,
Thank you for your answer. We only performed western but no Coomassie. We do not have that much blots with non-modified receptor (we currently do), however, the few we have disclose the predicted size. Thus, the inconsistency only occur with the tagged receptor. OK, modifications may be cell specific....
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