We were doing the monoclonal antibody generation. After the western blotting incubation with IRDye and infrared fluorescence detection. Two bands were found on the sample column of the membrane. The light band is much thicker than the heavy band. What is the reason behind? Because I thought the heavy chain should have a thicker band as its more bulky?
Thank you for your answer. So all in all heavy chain is more hydrophobic as compared to light chain. And because the light chain is more hydrophilic so it shows a thicker band on the membrane. I am afraid I still can't get the connection in between.
As Matthew pointed out, it's hard to know what you mean by "thicker"; I could not find a rigorous definition in my Scientific Terms dictionary :-) In any case, if you're talking about staining intensity, I am also wondering how you used IRDye. If you used an activated form for covalent binding, what kind of efficiency of labeling you get depends a lot on the protein amino acid composition. Even if you are doing non-covalent binding of dye, the efficiency of binding always depends on the properties of the specific protein. Since you are using monoclonals, potentially large differences between heavy and light chain amino acid composition are not averaged out by many species of antibody. Depending on how you are expressing your monoclonals, you may also be getting variable glycosylation of light chain, which would give a diffuse (thicker??), rather than sharp band.
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