I have expressed a fusion protein in E coli bacteria through simple induction method using IPTG and isolated the total protein from bacteria. Now, I want to check the thermal stability of the expressed fusion protein at different temperatures. Please suggest how I can check the thermal stability of this protein.
DEar Jyotsna Dayma
Do yuou mean, would you like to check thermal stability of the protein in the crude cell extract? with out purification?
Because after purification, you can easly assess the thermal stabilty of the protein using the DSF, which is simple. high trhougput and require low amount of sample
you can find information about DSF
https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu
determine the thermal stability in the extract is more difficult since you have the other proteins that can provide aspecific signals.
Theoretically id your protein is an enzime, and you have a specific activity assay, you can check as the incubation of the sample at different temperatures affect the activity, or otherwise in case that there are available some monoclonal antibodies that are reported to be able to recognize conformational epitopes of the proteins those are lost during the protein thermal unfolding you can try to set-up an ELISA assay with the aim to check how the ELISA signal of the protein change in function of the themperature.
However, i think that the simplest way is purify the protein from E.coli and afterwards use DSF to check is thermal stability.
Other approaches as:
- circular dichroism
- DSC
- 1D NMR
may also provide similar information, but i like a lot DSF since it is simple, cheap and require low sample amout.
good luck
Manuele
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