Has anyone recommendations for concentrating cell culture supernatants for ELISA?
Which cut off (5Ka?) would be suitable?
Cut off strictly depends on the size of the molecule you want to detect in the ELISA. It has to be considerably lower than the molecular weight of your protein of interest.
I would need more details about your experiment. whic is the expected concentration of your molecule in supernatants.ELISA is very sensitive, perhaps you dont need to concentrate the samples. For instance, hybridoma and cell transfection supernatants have to be diluted instead for screening.
I agree, cut off depends from the protein you are interested to keep. Also, serum concentration in culture could be an issue (10% serum become jelly if you concentrate a lot). My suggestion is to keep the cells in low (1-2%) serum for 24h and use those sup to concentrate. Also, very important is, in my opinion, to know the number of cells are in your culture at the moment of collection (not seeding). Number of cells influences the concentration of your protein dramatically and could lead to misinterpretation.
good luck
Michela
cutoff depends on the type of protein you are interested in. Have to tried ELISA with the concentration you have? I think with the kits now you can get result for moderate concentration also.
Hi everyone, thank you for all your replies. My conditioned media for ELISA is always low serum (2%). I have tried detecting soluble IDO-1 and PD-L1 proteins following IFNy stim for 24 h and I can detect them but just about (from media of a T75 flask). I could not detect anything in the same samples from a well of a 6-well plate, which would be ideal. So I want to take 1 ml of media from a 6-well plate (approx. 200K seeded, about 400K at time of harvest) and concentrate it so that I can detect something from the lower concentration of cells. The molecular weights of IDO-1 and PD-L1 are both over 40kDa but I assum their soluble products are smaller, closer to 20 kDa.
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