I just want to overexpression a peptides in hepatoma cells is just 15 kDa, but i get the postive protein is almost 60kDa. And the peptide could be binding with a 47kDa protein. I use the thermo fisher pCDNA3.1 and protocol and the cell was incubated with 1mg/ml g418.
Qiao,
You should consider other techniques to more accurately check the mass of your peptide, for instance, mass spectrometry. If you are not able to do that, try to do at least an RF analysis of your gel/blotting signal. I am presuming that your gel is under denaturating conditions. If everything is correct with your gel/blotting experiment I would guess that you have selected a clone expressing a concatamered plasmid, in other words, more than one copy being expressed in tandem. To check this you may sequence your construct and selecting other clones would be usefull.
I agree with the comments given by Newton. You need to confirm the correctness of your desired gene sequence from the expression plasmid. In addition, your peptide might have formed tetramers. You can try a sample buffer containing SDS and DTT for denaturing your peptides into its monomeric form prior to loading onto the gel. You need to also incubate cell lysates in the presence a sample buffer at 90-100 degree Celsius for 5 to 10 minutes. As suggested by Newton, using mass spec will give you more information to track the root cause of the problem.
I am agreed with them, I used the same vector with your lab and same antibody, and every time I checked protein size by Western Blot, I got the same size what I expected. You should check the sequence first. And secondly, with 15kDa, you can increase the percentage of SDS gels for small protein or use Mass spec for detecting. Furthermore, if you have protein for detection, you can compare the endo-lysis with the transfection-lysis.
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