I am using cell culture supernatants in an ELISA. The cells are incubated at 37C, centrifuged, then the supernatants are used in an ELISA at RT. Why does the supernatant collection need to be at 4C if all other steps are at RT or warmer?
Collecting at 4oC will stop protease activity, which could be significant if you have multiple samples to process. One will have little luck observing any real difference if the protein levels are further modified by unplanned degradation.
Remember that ELISA measures absolute protein levels, which means that any degradation will have a negative effect. Except you are interested in relative quantification.
We can't really do much about the cells needing 37oC to grow. You are fine once you start the ELISA, anyway.
Hello Natasha
You can definitely collect the cell culture supernatant at room temperature as well. There are no such rules that tells you that you have to collect your sup at 4 degrees temp. But during ELISA when you are blocking your samples with blocking buffer you need to incubate your ELISA plate at 4 degrees and this is same for primary antibody incubation (Just the same process like western blot). You need to store your supernatant at negative 20 degrees if you are planning to perform your experiment at any time in future. If you want to perform your experiment at any time on the same day of collection you can store your samples at 4 degrees too. The concept behind keeping your samples at 4 degrees is to reduce the degradation of your sample's proteins concentration. you can follow these protocols to gather more detail info:
https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/general-elisa-protocol.html#prot4
https://www.bio-rad-antibodies.com/helpful-elisa-hints.html
https://www.avivasysbio.com/protocols-procedures/elisa#ELISA
A temp. of 4°C for collecting cell supernatants is not necessary. It may reduce activity of enzymes in your sample. It may help to reduce possible quality-reducing influencing during harvesting when this process needs much time or when room temp. is higher that 23°C. However, this (RT) has no fundamental influence on your sample quality.
In any case, if samples were collected at same conditions (4°C or RT or what else), results are comparable among each other. And this is the most important point in your experimental setting.
- Badges
- Science method