Protease Assay - (Jul/12/2010 )
Hai All
Iam working on extracellular product of mammalian cell culture. Hence, I would like to quantity the proteases that degrade my target protein. I used colorimetric protease assay kit from pierce thermo Scientific but I didnt get any results. It seems like there is an interaction between my samples and TNBSA (trinitrobenzene Sulfonic acid). So, Iam using Florescent protease assay kit from the same company. But I dont get any results.
Iam wondering, is there no proteases inside e sample bcoz I did get the correct standard (Trypsin) values in the assay. If there is something wrong in my analysis, How to analyze fluorescent based protease assay?
Else, I need to use other type of quantification of proteases?
Your comments and Suggestion on this matter is highly appreciated.
thanks in advance.
mouli on Jul 13 2010, 06:53 AM said:
Iam working on extracellular product of mammalian cell culture. Hence, I would like to quantity the proteases that degrade my target protein. I used colorimetric protease assay kit from pierce thermo Scientific but I didnt get any results. It seems like there is an interaction between my samples and TNBSA (trinitrobenzene Sulfonic acid). So, Iam using Florescent protease assay kit from the same company. But I dont get any results.
Iam wondering, is there no proteases inside e sample bcoz I did get the correct standard (Trypsin) values in the assay. If there is something wrong in my analysis, How to analyze fluorescent based protease assay?
Else, I need to use other type of quantification of proteases?
Your comments and Suggestion on this matter is highly appreciated.
thanks in advance.
Do you have any serum in your samples? This contains many protease inhibitors that could mess with you assays
there are a lot of different classes of proteases. there is no one simple assay for all of them. nor is there one substrate suitable for all proteases.
we assay for a couple of different proteases using 14c-casein as a substrate. azocasein could also be used but was not sensitive enough for our purposes.
other potential general substrates include hemoglobin, bsa, etc. just about any ubiquitous protein.
some proteases require low pH, high pH, neutral pH, calcium,...
if you are looking for a specific protease or class of proteases then you need to check the literature for the proper assay.
klinmed on Jul 13 2010, 10:26 PM said:
mouli on Jul 13 2010, 06:53 AM said:
Iam working on extracellular product of mammalian cell culture. Hence, I would like to quantity the proteases that degrade my target protein. I used colorimetric protease assay kit from pierce thermo Scientific but I didnt get any results. It seems like there is an interaction between my samples and TNBSA (trinitrobenzene Sulfonic acid). So, Iam using Florescent protease assay kit from the same company. But I dont get any results.
Iam wondering, is there no proteases inside e sample bcoz I did get the correct standard (Trypsin) values in the assay. If there is something wrong in my analysis, How to analyze fluorescent based protease assay?
Else, I need to use other type of quantification of proteases?
Your comments and Suggestion on this matter is highly appreciated.
thanks in advance.
Do you have any serum in your samples? This contains many protease inhibitors that could mess with you assays
Thanks for the reply.
Yes, my samples are from mammalian cell culture of DMEM Media with 2% serum (FBS).
So, in that case, how would I estimate the quantity of protease concentration?
mdfenko on Jul 13 2010, 10:38 PM said:
we assay for a couple of different proteases using 14c-casein as a substrate. azocasein could also be used but was not sensitive enough for our purposes.
other potential general substrates include hemoglobin, bsa, etc. just about any ubiquitous protein.
some proteases require low pH, high pH, neutral pH, calcium,...
if you are looking for a specific protease or class of proteases then you need to check the literature for the proper assay.
oh okay. Thanks for the information, bro.
my samples consists of serum proteases and proteases from mammalian cell culture. what are those proteases normally? any ideas bro?