I have purified a monoclonal antibody from a hybridoma solution, and when I runn it on an SDS-PAGE gel I get 2 bands for the light chain. I know that an antibody is 150 KDa and when we boil it in the presence of DTT and run it on an SDS-PAGE gel we should get 2 bands, one for the heavy chain at 50 KDa and the other one for the light chain at 25 KDa (An antibody consists of 2 heavy chains and 2 light chains).
Though we can get 2 bands for the heavy chain (as it can get glycosylated), what is confusing me now is that I am getting 2 bands for the light chain (the difference between the bands is not more than 2-3 KDa).
Can the light chain of the antibody be glycosylated? Or is the antibody getting degraded (it should not as I store in NaN3 and I purified it at a couple of months ago).
Can anyone please help me try to find the reason for getting 2 bands for the light chain of the antibody on the gel?
Many conditions can affect SDS-PAGE. And the most common factors are salt concentration and the pH. I am not sure how you purify your antibody but if you use affinity column, the sample generally has high salt concentration or low pH. So neutralize the pH or decrease [salt] and see what will happen.
I would say " couple of months" is enough even for stable antibody to start degrading. I would try again with freshly prepared sample. What is the purification strategy? Is it protein A purification? And is it stored frozen or at +4?
One question ahead: at which molecular weights you find the L-chains?
Don believe that hybridomas produce complete IgG only. You can find any permutation of composites of heavy and light chains. Do visualize them, we used a western blotting with a double staining and anti-Kappa/anti-Lambda-AP (substrate: BCIP/NBT) and anti-gamma-HRP (substrate: DAB + NiCl2).
Interestingly we found
L1 (probably no reaction with your antigen)
L2 (can recognize your antigen)
H1L1 (will recognize your antigen)
H2 (can recognize your antigen)
H1L2 (will recognize your antigen)
H2L2 (will recognize your antigen) (the only complete IgG)
See also: Kießig, ST, Marx, U, Wilke, B, Hausdorf, G (1991) Immunoglobulin fragments during fermentation of a human monoclonal anti-erythrocyte antibody, ESACT Meeting, Brighton 1991, S 38
http://www.sciencedirect.com/science/article/pii/B9780750604215501331
IgG can be glycosylated on the light chain; it's not as common as heavy chain glycosylation. If you want to double check, you can do a lectin blot. Something that recognizes mannose or sialic acid residues would probably be your best bet. I've attached a paper where they saw some MW changes in light chains that might be helpful.
Hello All,
I am also experiencing the same problem with my purified polyclonal IgY antibody. attaching the figure below. why there is multiple bands other than the heavy chain and light chain bands.
I feel this is simply affinity purification of your Ab. I depends upon the type of kit or matrix you have used. Affinity purification generally yields products with some contamination (May be 70% purity). Other bands you are getting are due to non-specific binding to the matrix. If you want to remove these contaminants from your preparations you may use size exclusion chromatography. It will yield the desired Ab with more than 90% homogeneity.
Could anyone find an explanation for two bands observed at the light chain? I have the same problem after purification of chicken IgY from egg yolks with a commercial kit. There is a dispute about the size of the light chain. Some literature states that the size is 22 kDa (the kit as well) and some say the light chain is 27 kDa. However, I found both sizes?
hello Mariëtte Ferreira and Jobin Jose Kattoor can you please give me the details of your gel % and sample loading buffer???...... because the problem with mine is i am not getting light chains when running IgY......... i am using 1% beta mercaptoethanol in my loading buffer.... is that cause any effect on igY??? kindly suggest me your details.....
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