RIPA lysis buffer composition

James B. Hutchins jbh at anat.UMSMED.EDU
Sat Oct 30 11:43:30 EST 1993


In article <simmenlab.10.751917998 at animal.ufl.edu> simmenlab at animal.ufl.edu
(Dr. Simmen's Lab) writes:
>
>I am currently following a protocol that uses a buffer termed RIPA 
>lysis buffer.  The buffer is currently being used in the preparation of 
>conceptus homogenates which are subsequently assayed for DNA content and 
>protein concentrations.  I have a suspicion that our recipe for this buffer 
>is incorrect.  Does anyone know if this is a commonly used buffer?  If so, 
>what is its composition? Specifically, the problem appears to be the Triton-
>X 100 concentration of the buffer which interfers dramatically with our DNA 
>fluorometric assay.  Any comments or suggestions are welcomed.
>
>					Thank You,
>					Mike L. Green
>

Mike,

Here is the recipe from our lab (from the Big Red Book, aka _Current
Protocols in Molecular Biology_, Ausubel et al., p. 10.16.8):

1% sodium deoxycholate
0.1% SDS
1% Triton X-100
1% bovine hemoglobin
1 mM iodoacetamide
  (protease inhibitors: they list aprotinin and PMSF; we use aprotinin,
  leupeptin, AEBSF (PefaBloc: Boehringer), EDTA, pepstatin)
10 mM Tris.HCl pH 8.0
140 mM NaCl
0.025% NaN3

However, I would bet that almost any detergent autofluoresces.  That seems
to be what you're referring to as far as 'interference'.

Anyone out there who can suggest whether this is the case, and if there
is a non-fluorescent detergent Mike can use?

Jim

-- 
Jim Hutchins                    []     E-Mail: jbh at anat.umsmed.edu
Asst Prof of Anatomy            []     Asst Prof of Neurology
Univ Mississippi Med Ctr        []     Jackson, MS



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