I wanted to use proteinase K to treat conditioned media in order to determine if a protein factor is responsible for the effect seen when CM is used on cells. I have come across various protocols in the literature. Does anyone have experience in using proteinase K for this purpose? I am trying to find the best way to determine if the PK is working. I have treated with 200ug/ml and neutralized with 5mM Pefablock but then when using this on my cells they all died. I think the Pefablock lowered the PH of my conditioned media. I then tried using 10ug/ml followed by heat inactivation but I need to find an assay that will prove that this concentration in my conditioned media actually degraded the proteins. Any advice will be appreciated.
Proteinase K, is produced by the fungus Tritirachium album Limber, is a serine protease that exhibits a very broad cleavage specificity. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples.A typical working concentration for Proteinase K is 50–100µg/ml is added to treat conditioned media.
Thanks Ravi,
Do you think heat inactivation of proteinase K or treatment with Pefablock will be best if the conditioned media will then be used to treat other cells?
You wanna heat inactivate before you can treat your cells. being an enzyme it may digest some of the membrane proteins of the cells. thanks
Dear Jeremy, did you solve the problem at last, I also encounter the same problem and if it is possible you tell me what did you do? Thank you so much, any response will help a lot.
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