A glycoprotein in two breast cancer cell line was tested by western blot, and one band was probed in no.1 cell line, while four bands were probed in no.2 cell line. Moreover, the MW of band in no.1 is different from any of the no.2. so i want to know the reason, just as the different modification or some procedure i neglected during WB? THANKS ANYONE FOR ANSWER.
Hi Tianli,
proteins could had differents (and multiple) glycosylation sites and different sets of glycosiltransferases are expresed depending on the cell line, resulting in several WB profiles. I also recomend you to test the secondary antibody (or similar reagent depending on your detection system) for unespecific recognizing. May be it would be usefull if you give me more information about the type of glycosilation, glycoprotein and conditions of detection (i.e if you are using antibodies, how are you blocking, etc).
Good luck
Thank you Romina, this glycoprotein has two glycosylation sites in N-terminal, and i use a flag antibody to test the ectopic expression in the cell. no more secondary antibody to flag tag i have. and nonfat dried milk powder blocking buffer was uesd as usual. could you give me more advise, or anyone? thanks again.
When proteins are modificated in Golgi, in general the glycosylatyion site/s are extended. Probably each cell line is expresing a different set of enzymes and this is the reason why you are not detecting the same bands in each condition, because there are not the same chains of suggar. To check unespecificity in this case, you has probably verified the non transfected condition (you should not detect a band when using anti-flag).
@ Romina, yes, i see what you mean. is there any tips when abstracting glycoprotein in the cell, just different from nomal proteins? Thank you!
In our lab we usually work studing o-type glycosylation. In general terms you should work it like a normal protein (if you are going to extract it is important the subcelular location and if it is soluble or a membrane protein). Then the protocol could vary to detect glycosylated terminals, but in your case you are detecting the protein (no the PTM). I hope this information could be usefull for you
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