Hi,
I have one question for my experiment. I’m making single copy inserted worms using MosSCI method.
For this, I have constructed vectors containing with promoter(3.5kb)+CDS+3XFLAG+3’utr(my gene’s).
After injection, I isolated inserted worms using PCR. I had no problem so far.
BUT, when I tested protein expression for single copy inserted worms, I couldn’t detect my flag tagged protein through immunoblotting by anti-FLAG antibody.
My protein is well expressed in transgenic worms (extrachromosomal array) but not inserted worms.
What can I do to solve this problem? I need your help.
Remember, it’s single copy so the expression level will be markedly lower (10-100x). Are you sure you could detect that amount? You could also try making a GFP fusion and see if you can detect fluorescence. In my experience, soluble mCherry expressed from a MosSCI insertions, even from a promoter that is fairly strong by high-copy transgene (<5ng/ul injected, easily seen by a dissection scope), is barely detectable at single copy even under a compound epifluorescence microscope at 100x.
I would recommend trying a 3xFLAG IP against your strain vs. a control to see whether you can enrich your protein to levels that are detectable by WB. You could even try this as a denaturing IP (solubilizing in 1% SDS and then dilute up in RIPA buffer minus SDS). Do it in parallel to your over-expressing strain.
That’s right.
using amount of worms for IP is a way to enrich the single copy protein. for one of our single copy strain, its anti-FLAG western is quite weak while IP result is strong.
