Probably, low plasmid purity affected your transfection efficiency and resulted in low protein expression. I dont think it is protein degradation since you dont see any degradation products in your membrane. Also, if your inserted tag is not in the epitope region of the antibody, antibody/protein interaction should not be affected.

Good luck

Miray Turk

Thanks for your answer. Could you tell me more about 'degradation products in your membrane'? If the protein got degraded, what would I be supposed to see and does it depend on the antibody? I am asking because we were expecting protein degradation but right now we cannot rule out low protein expression due to bad plasmids.

Our inserted tag is on the N and C terminal, and the epitope of the antibody is in the middle, at least 50 aa's away from the terminal.

Right now we found these plasmids might have grown faster. So the plasmid quality may be an issue due to cell death. With 30C culture we were able to increase the plasmid concentration and quality (by a bit, not too much).

Hello,

If your protein is getting degraded in the cell, you should observe additional protein bands smaller than your target protein in your WB membrane. But for me, your main problem is low plasmid quality which affects transfection efficiency and probably cell viability. It looks ethanol is not properly removed from sample during plasmid isolation.

Good luck

Hello, are you extracting your plasmid with a miniprep kit or any silica column-based commercial kits?

If you are, I will do a maxiprep instead, which although it takes two full days, you will get a decent plasmid concentration (over 1mg/ml) and transfection-quality purity. Hopefully sorting out your issues in the downstream applications as well.

Best of luck!

Thanks for the answers so far. Yes, our current protocol uses predominantly the miniprep kit from Promega. I have not used the maxiprep kit but if we decided to diagnose plasmid purity as our next step we will certainly take it into consideration.

The cells grown in 30C showed good plasmid concentration (300 ng/uL) but the A260/230 ratio was still questionable, and the protein expression did not improve in these newly made plasmids.

So far it does seem the plasmids did not transfect well. Last week we ran qPCR on the vector +/- insert and some of our vector + insert showed the same level of transcripts as the original, but the ones with questionable quality did not show good transcription. We're now re-transforming the cells from the PCR reaction since the last time the ampicilin stock was starting to go bad which might have resulted in the bad plasmids (although they did sequence well).

I would recommend fellow researchers to use qPCR to correct for some effects of transfection at least once if anything seems questionable.

Hello everyone,

I think I have an answer to this situation, it's likely the LB media we used for liquid culture was bad, possibly because it was contaminated or I missed adding a component in it. I made a new batch of LB media and the vectors of interest grew decently with good quality plasmids extracted. I did a western blot recently and it seems the expression is now present.