I assume you have the plasmid that overexpresses your protein of interest in the target cells and when you conduct the WB of the lysates with anti-Flag antibody you detect flag signals at the 55kd, but your protein of interest predicted size is 60kd? And you don't have a specific antibody for the protein of interest. Well, it might be just an actual size of your target to be 55 kd. Have you tried to order recombinant protein of interest run it on the gel and detect it with the Coomassie staining or Puancro red staining? If you get 55 kd band with Coomassie from recombinant protein that is its the actual size in the gel. The variations of protein mobility on the gel from predicted size happen quite often and not related to protein degradation.

Anton Lennikov I am sorry I did not explain it vey well. We have specific antibody for the protein. But the antibody did not react with the protein we made. At the same time we also bought the protein overexpressing cell lysate. When we use the commercial cell lysate to do WB, higher molecular band(around 61KD) is showen. The commercial overexpressing cell lysate reacted with the specific antibody.

Dear Zhang Xu,

the antibody is specific for a distinct and reachable peptide sequence (often 5-7 peptides). Maybe - and if you overexpressed a correct AA sequence of the protein of interest using your DNA construct- the "specific" antibody may not reach the correct peptide sequence (due to folding effects of the construct different to folding of the native form of the protein).

Another possibility is, that your "specific" antibody is not specific (e.g. see for ref. Baker M. Reproducibility crisis Blame it on the antibodies. Nature. 2015_521.p274-6. doi 10.1038521274a).

However, since the protein detected is smaller that the expected size of the protein of interest, I think that the product - corresponding to you overexpression sequence - is not the same as the native protein (maybe due to misfolding, different post-translational processing, or due to an incorrect (c)DNA construct).

Do you detect flag tag in your construct overexpressed lysate from HEK? Since it will allow you to tell if the protein produced at all or not. If you detect the flag something might affect the antibody binding due to construct architecture or added fusion flag. Maybe something is missing from the construct sequence producing partial protein (that would explain smaller size). Another possibility is that the antibody may not be that specific as mentioned by Andreas Eisenreich , you should try the other antibody strain. Preferably polyclonal.

Andreas Eisenreich Many thanks. I have checked the sequence of my plasmid and it looks ok (I could just get the results from one primer, the primer for reverse side of the sequence couldn't work well ). If there is misfolding or post-translational processing, may I ask some suggestions for improving the experiment? By the way, my protein is a kind of N-glycosylated protein.

Anton Lennikov Thanks s lot for your answering. I detected the flag tag and I got three bands: around 125kd, 55kd, 51kd. I attached the picture here. From left side, Marker, protein overexpressing cell lysate, negative control respectively

This strongly depends on the nature of your protein (human, insect, bacteria), maybe the species and, therefore, the type cells used for adequate expression and processing of your rec protein. I would propose to check methodological information from corresponding literature.

Have you checked proteolysis possibility?

There are many factors that can affect the (apparent) MW in SDS gel electrophoresis: e.g incomplete unfolding, phosphorylation, proteolysis. Are you reducing your sample? Disulfide bridges also can affect the mobility. If your protein is glycosylated, the expression system may influence the size of the sugar moiety. Mass spec would show the real MW, and if glycosylation is a factor, you might try to compare the deglycosylated sample and positive control.

The end product prrotein has to be prepared in sufficient quantity. Then the possible primer sequence, post translate modificatioinvestigatedns has to be analysed.

There is some stability problem here or you are not expressing the protein you think. Your protease inhibiting cocktail might not be suitable. You might use PMSF for the inhibition of proteolysis, it is not better but cheaper and has different specificity. You might also cut the band off the gel and send for LC-MS identification, it is cheap nowadays, to confirm that your 55k is what you are looking for or just some random rubbish...

Many thanks for all the answers. I have learned from your suggestions. Could anyone give me some suggestions if it is because post translate modificatioin (misfolding of glycosylation)? In this case, what should I do to get the right target protein? By the way, I also used PMSF in the experiment.

Zhang Xu

There may be some reasons as follow.

1. Recheck your construct and protocol for your recombination.

2. I don't think it will create much difference between 55 -60 KD.

3. If your protein is okay, then check the WB procedure that where are you lacking.

To detect whether proteolysis is present, the easiest way is if your protein carries detectable tags on either end, e.g. N-terminal FLAG and C-terminal His-tag: If your product underwent cleavage, one of the two detection reagents would fail to stain the band. To test for glycosylation, you would test whether the mobility changes if you pretreat your sample with glycosidases (e.g. https://www.sigmaaldrich.com/technical-documents/articles/biofiles/glycoprotein-deglycosylation.html?gclid=Cj0KCQjwoaz3BRDnARIsAF1RfLexowyKtsTL_8ydiXbdCXjZCr3Wjr5SOcMVxoatYP4Ba2uQDA1NSswaAv7UEALw_wcB). Dito for phosphorylation: pre-treatment with e.g. alkaline phosphatase would change the mobility. For incomplete denaturation, you test by boiling your sample for different times in the SDS loading buffer, +/- mercaptoethanol to test for disulfide bridges.

Mass spec of the whole molecule would test for any covalent modification, and the difference between the expected and the observed mass would give an indication of the potential modification. Mass spec after proteolytic digestion would indicate whether the band is indeed derived from the expected protein, or whether it is a different protein that crossreacts with your detection system.