I'm modifying the ELISA kit assay to detect the secreted protein in the cell culture in under a defined media. Based on the instruction on the kit, typically they would follow by sonication of the cell pellet in PBS and extract the crude protein in the supernatant layer.
But I would like to more focus on the secreted protein in the cultured media, as I don't know the concentration of the secreted target protein with tag-fusion. Can i directly culture the cell in the ELISA well then followed by the wash and visualization? i don't mind the interference of the living cell since i have an empty vector control but i am just a bit skeptical about the interference of directly applying the cell culture in the reaction. Any thought?
Dear Hsiao-Chun,
I wouldn't culture the cells in the ELISA-plate, as the bacteria probably will destroy the coated capture antibodies, and also will bring in peroxidase activity, and in this way will cause high and uncontrollable background.,
But, instead of the sonicated cell pellet, using the supernatant (cleared by a sharp centrifugation or better with a 0.1uM syringe filter or by spinning through such a membrane) should do the trick.
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