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| Hi, I am working with an unknown protein. The calculated size should be about 70 kDa. I produce it in SaOS-Cells and it is secreted into the culture medium. It has a his and a myc tag. When I analyse it on SDS-Page and Western Blot I always get a big and clear band at about 100 kDa and only a very thin band at 70 kDa. I know that inside the cell it has the right size but after the secretion it is 30 kDa larger. I already had it in MS (tryptic digestion) and only my protein and no other protein came up. So what can it be that is bound to my protein? We already discussed glycosylation. But people told me for that the band is to clear. We checked with a glycosylation inhibitor and the protein is only about 5 kDa smaller. If you have any idea what I could check for, that would be great. Thank you very much. |
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