I have a membrane protein with a GFP tag on its C-terminus. I have been trying to perform a co-ip on this protein by pulling on the GFP tag, but it has come to my attention that the GFP tag may be being cleaved off of my protein. Is there a way to prevent such cleavage in the HeLa cells I am working in?
Charles, see Tammy Pracheil · Tulane University answer to a similar question concerning the function of GFP tagged Proteins.....
"I've done extensive tagging with GFP and yes it can interfere with function. To minimize interference, I take into consideration whether there are domains at either end of the protein, such as a C terminal tail-anchor domain which can be identified by looking at a hydropathy plot of your protein and avoid tagging at that end...then I always include a "spacer" typically a 10 alanine linker between the protein and the tag, and lastly if functionality is critical I must have a way of confirming functionality after tagging, i.e. growth defect recovery or some other test for function. If there is no known way to test functionality but I've considered the two other things above, I just be sure to test several clones rather than rely on just one to be sure the results all agree."
Thank you Raouf for that answer. I know the effect of GFP on my protein, however I am unclear how to prevent it from cleaving off. My construct was made about 10 years ago and has an 8 amino acid linker. I am unclear if this cleavage is a recent problem or if other people have had it. When I straight out lyse the cells in sample buffer it becomes visible on the westerns. I am wondering if the HeLas are producing the cleavage product or if it occurs during my lysis. What are your thoughts?
since the cleavage is occurring after you had lysed the cells, it is most likely due to the release of enzymes during the cell lysis process.... If the HeLas are causing the cleavage you should have seen it on your western plots before or after the cell lysis.
In this case try adding different enzyme inhibitors to your cell lysis cocktail to see if you can inhibit such enzymes from causing the cleavage in your cell lysate.
Sequencing the construct will tell you if a cleavage site was engineered into the construct. Does it happen in other cells (293, cho), also add protease mix to your lysis buffer.
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