CAT assay problem!

jr at jr at
Wed Mar 15 16:59:54 EST 1995

Dear All,

               I've just started to do CAT assays on transfected HeLa cells 
after a lay off of a few years. The overall assay is similar to that described 
in Maniatis. The cells are washed twice with cold PBS, scraped into 1ml PBS, 
spun down and resuspended in 150 ul of Tris-HCl (pH 7.8) and subjected to 
three rounds of freeze/thaw. Cell debris and nuclei are spun down in a 
microcentrifuge tube and the supernatant heated at 65C for 15 min before using 
in the CAT assay. The problem I have is that after heating the cell extract 
for 15 min at 65C, the extract "goes cloudy" and the ppt can be spun down.  My 
question is what is this ppt. and will it interefere in the subsequent CAT 
assay and can I prevent it coming through. Also, are there any ways of 
increasing the protein content of my cell extracts. I seem to recall that 
detergent in the lysis buffer interferes with the assay itelf.

                  Thanks in advance.


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