Mammalian Lysis buffer for B-gal Assay
preben.lexow at student.uib.no
Thu Sep 9 08:40:50 EST 1999
"J.A.H. Hendriks" wrote:
> Mimi Liao <m.liao at student.unsw.edu.au> schreef in berichtnieuws
> 188.8.131.52.19990820122254.00795a20 at pop3.student.unsw.edu.au...
> > Hi all,
> > I am currently lysing the CHO-K1 cells with lysis buffer containg 50 mM
> > Tris-Cl (pH8.0), 150mM NaCl, and 1 % Trioton-100. And i'm measuring the
> > B-gal content using ONPG colormetric assay. when i added the Na2HCO3 to
> > stop the reaction, the solution became very cloudy. However, the solution
> > cleared up o/n.
> > My questions are;
> > 1) Do we really need to add the stop solution ? Can we just measure the
> > absorbance straight away without stopping the reaction ?
> > 2)Are there any recipe for mammalian lysis buffer which are compatible
> > B-gal assay ?
> > Thanks very much in advance
> I allways do a chloroform extraction and a 10 minute spin after stopping the
> color-reaction. Everything has to be done in the same way and I think this
> would be very hard if you don't stop the reaction.
> Good luck
The reaction is a function of time. So if the samples are meassured after
different time delays. The assay would be completely wrong. Another problem is
that after a very long time the product of the reaction will be the same in all
test tubes. You will not be able to meassure the diffrence in colour
More information about the Methods