Hi All,
I have performed Non-Reducing SDS-PAGE for a pure antibody where I have observed multiple bands instead of a single intact band. Kindly suggest the probable reasons.
Firstly, could you tell how you made your antibody and how you purified your antibody?
Yes it is a glycoprotein.
Purification is done by regular purifications steps involved in DSP and we get the final product which is in a stable formulation buffer.
https://home.ccr.cancer.gov/lco/ImprovedMaturation.htm
Please check thelinks, if it helps.
We made our fabs by overexpression in 293F cells. We also see extra band, which is believed to be one heavy chain without forming fab with light chain. It will be of great help if you could clearly show how you did your experiment.
I agree with Zhang.
depending on your purification system, you can expect a heavy chain (50kda), light chain (25 kda), a combination of 1 heavy and 1 light chain (75kd) and complete antibody (150kd).
Different levels of glycolysation may also result in multiple bands. This problem occurs when cells are not completely separated from ab containing supernatant. Cell lysis during fermentation also may create such problems.
Can we have a look at the gel?
Hard to say what the different bands are without the molecular weight of the three proteins.
Remember, Molecular Mass of glycoproteins will be not be accurate in SDS-PAGE. Check papers from Leach, Collawm and Fish from 1980 in both Analytical Biochem and Biochem journals.
One thing that can be happening is the SDS is not binding to the protein well so you are getting various amounts of charge and thus mobility, Remember that any glycosylation will not bind SDS thus changing the charge to mass ratio of SDS to protein.
Thank you all.
These multiple bands are observed in between 100 and 150kDa, apart from the intact band at 150kDa. So my query is what could lead to such extra bands in between 100 & 150kDa. Please find the gel below for your information.
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