Mammalian Lysis buffer for B-gal Assay

Preben Lexow 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
> 3.0.1.32.19990820122254.00795a20 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
> with
> > 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
> Brenda

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
intensity!!!

Anders.





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