I used a commercial kit to purify IgY from chicken egg yolks. According to some literature and the kit's manual the light chain should be 22 kDa. According to other literature the light chain is 27 kDa. Why do I find both sizes in all my samples? Are these only impurities or is it perhaps due to glycosylation of the light chain?
I expect it is due to glycosylation difference. Check if there are glycoslyation sites on the protein sequence.
Hi Mariëtte Ferreira ,
Purified monoclonal antibodies are known to show two distinct bands (Light chain and Heavy chain) under reducing SDS-PAGE conditions. However antibodies purified using kits are known to show more than two bands under reducing conditions.
In case of IgY; one should expect heavy chain, light chain, truncated form of heavy chain (reported for IgY) and heavy chain fragment.
Glycosylation usually occurs in the Heavy chain; but if you suspect it could even be glycation (not glycosylation) of the light chain.
Try to do a PAS staining/ lectin blotting of the SDS-PAGE; you will come to know whether the light chain is glycated/ glycosylated.
The best way is to cut/ excise all these bands, trypsinize and get it identified using a Mass spec; so that you will be able to authentically identify each of those bands.
Hope this helps....
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