This analysis was meant to verify the presence of my recombinant protein that is transiently expressed in Nicotiana benthamiana. The transgene consists of a chloroplast targeting peptide (CTP) and the N terminal and a His-tag at the C terminal. For expressed protein still consisting of the CTP, the expected molecular weight is 25kDA whereas for proteins not consisting of the CTP, the expected molecular weight is 18kDA. However, aside from acquiring these two bands on the gel, there are three other bands formed which lie between 18kDA and 25kDA. Can anyone explain this scenario?
Sezer Okay
The antibody that i used was a monoclonal antibody. I have been thinking that it might be due to degradation but i can't seem to find exactly what protease is the one within the plant cell that is causing the degradation. I have tried using proteolysis cleavage software to predict what enzyme that is causing this but to no avail.
Do you think that such degradation could be due to the protein extract being stored for too long? After the protein is extracted, i kept them in -80 degree celcius fridge and freeze-thawed them twice before performing the western blot and immunoblot..
So you are suggesting that the proteins could be degraded during the extraction procedures?
Could you retype the last sentence as i don't quite get what you mean.. sorry..
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