Hi everyone,
I have been trying for months now to get my protein expression confirmed by western blot and failed every single time (I checked the antobodies, they are working fine). I have transformed yeast (Pichia pastoris KM71H) with my plasmid by eletrocporation (4 kb long, my protein is 26 kDa, 1210 bp) confirmed transformants via PCR but I don't get any protein produced afterwards. Does anyone have any idea on what is going on? Is it possible that my confirmation on PCR is actually a false positive result?
If someone could help me with that it would be awesome, thank you in advance!
How are you using the PCR to check for positives? I assume you are streaking out potential positive colonies on a NEW plate & not doing colony PCR from your original selection plate. Colony PCR is notorious for false positives if you use the colonies on the original selection plate.
What promotor are you using? Inducible or constitutive? Is the insert in the correct reading frame? Is the protein toxic?
Lots of possibilities. What else have you tested?
Hi there,
Are you 100% sure of the plasmid construct?
PCR screening requires different controls: PCR run on the untransformed cell, PCR run on the cell transformed with an empty plasmid. Only comparison with those controls will tell you the relevance of what you get with transformants.
Is your protein to supposed to be secreted or not? (I'm puzzled about the size of the DNA and the size of the protein...)
Is expression supposed to be inducible or constitutive?
If you are doing colony PCR try performing negative controls on untransformed cells
What promoter did you use, and which inducer??
Another suggestion may be the targeted protein is toxic to yeast cells and cause their death?
I suggest you confirm your insertions cloning with Sanger.
Also, if I were in your, before doing the Western blot, I would measure the protein band using Coomassie blue staining on the SDS-page gel.
I highly recommend using a different IgG, in my case our ab recognized a portion that may have been internal (hydrophobic), the switch to another IgG that recognized a different peptide was the solution.
Also, try to run your samples with Guad for (solubilizing) mixed in them, your POI may be stuck and not running into the gels.
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