Dear All,
I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on the N-terminus. Roughly about 12-18 hr after transfection, I observe the GFP signal in the cytosol, however, the signal is very low compared with pCDNA3.1-expressing GFP alone plasmid. Also, by about 48 hr, the expression diminishes further and thereafter it is barely visible by the fluorescent microscope. However, the GFP alone pCDNA3.1 construct expresses very well. I switched the vector backbone to p-N-EGFP vector and I even used the N174-MCS-Puro lentivirus plasmid. In the the different backbones, I observe the same situation with my protein of interest. What could be the reason? Has anyone observed this with another cytosolic/membrane proteins?
Thanks for your help.
Regards.
This situation is quite normal when I fused GFP with my interested proteins. The reason may be various, such as if your protein affects GFP folding, then it can not function as well as GFP alone, or if your protein was degraded quickly somehow. If I were you, I would fuse the protein with a small tag ( like HA, FLAG, or Myc), then do ICC with an anti-tag antibody to verify the protein localization.
Dear Nam, thanks for your point. I forgot to mention that I tried using FLAG as well. Although, I didn't do ICC, I did perform sub cellular fractionation and WB. There as well, the expression of FLAG-tagged protein was barely detectable compared with another FLAG-tagged protein.
Maybe your protein of interest has a quick turnover kinetics, i.e., it gets degraded by the cell within 20hours. As for the difference in signal intensity between empty vector and fused protein, I have also observed the same with my protein of interest. I think its due to the difference between transfection efficiency, nuclear import kinetics, transcription and translation efficiencies of the GFP plasmid and your transgene. Try to conclude your experiment within 20h.
All the best.
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