I would like to know what you use for expression controls of destination gateway vectors for example in IP or transfection.
Let me illustrate with an example, for example you want to express pDEST-GFPMYPROTEIN and perform an anti-GFP-IP. Classically you would use the empty vector to express GFP alone and then compare what you got with your GFP-MYPROTEIN.
However it's not possible to properly express empty gateway vectors due to all the stuff there is in the gateway K7...
For GFP IP I have used a pEGFP C1 vector as control, even if it's not exactly the same as the GFP gateway vector backbone I have. However for other tags (V5, HA...) I don't always have a decent control. I wonder if anyone has tried just to do an entry vector with some stop codons or a small stretch of amino acids so that you could recombine with your DEST vectors to be able to express them empty?
Hi Daniel,
what I did in a similar case, was to clone a pEntry-GFP vector and gateway-cloned that in my pDEST to have exactly what you described: a control plasmid with pDEST-GFP-only plasmid expressing GFP plus all tags that come with the pDEST.
Then you IP your GFP-fusion and compare it directly with GFP-only IP. The difference should be your specific interactors (excluding the once that may just stick to GFP).
Best wishes,
Andreas
Hi Daniel,
I agree with the above post by Andreas. Nevertheless, if I understood your question correctly, you would like to have a control in the context of a pDEST plamid that produces fusion proteins with a GFP tag already encoded in the plasmid backbone outside the Gateway cassette. In this case, you could still use the pENTRY-GFP vector that Andreas mentioned, provided the GFP in it still had a STOP codon. Otherwise you would end up with a tandem-dimer GFP fusion being expressed.
Good luck!
Tomas
Thank you both, it's true that for an N-terminal GFP destination vector you end up with a tandem GFP product (which is my case)... But I'm also interested for other tags and this ends up being good and traceable control I guess (V5-GFP, HA-GFP)
Hi Daniel,
I just realized I had misunderstood your initial post. I see now that you are using fusions with the GFP or other tags at the N-terminal end of the fusion protein. For the expression of your GFP-only control, It may be worthwhile checking the sequence of your original DEST vector for the presence of any stop codon in the intact Gateway cassette just downstream of the sequence in common between the attB1 and attR1 sites. Since the att sites are designed so that they remain in-frame with whatever is cloned afterwards, there might be one in the Gateway cassette of your vector shortly after the attB1/attR1 common sequence that may cause GFP to end with a little tail that may cause no interference if expressed from the original DEST vector. I did a quick check on a vector I have (pEFDEST51) and I found a TAA sequence in frame with the attB1/attR1-shared sequence. Since the Gateway casseettes are pretty modular across the pDEST vectors, ther may well be one in your vector too. In any case, beware the Gateway cassette will add a long 3'UTR till the polyA signal your vector uses. All in all, it means you might be able to transfect your non-recombined vector and get a GFP-only protein as a control. I hope this helps!
Tomas
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