IUBio

Inhibition of luciferase by bood

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Mar 17 21:28:50 EST 1999


In article <mheiges-ya02408000R1703992022160001 at news.uga.edu>,
mheiges at cellmate.cb.uga.edu wrote:

> In article <whc23-1703990834040001 at powercenter-604.physiol.cam.ac.uk>,
> whc23 at cus.cam.ac.uk (Dr.W.H. Colledge) wrote:
> 
> > Does anyone know if the presence of blood in a lysate will interfere with
> > a luciferase assay and why? Thanks 

> In my experience, there is a little interference. If I remember correctly,
> maybe as much as a 5-fold to 10-fold reduction if measuring in straight
> blood + lysing buffer (well, I guess that qualifies as "alot" of
> interference). I presume it's because the reddish heme is reducing light
> output. Washing the cells to remove the serum has no effect for me so I
> don't think the serum interferes. And if I reduce the number of RBCs my
> assays improve.
> -Mark

Its also possible to see luciferase activity from
protein produced by in vitro transcription/translation
(eg. Promega TnT) using a rabbit reticulocyte system
which is necessarily full of haem.  I've never done
a direct comparison of the amount of protein (eg. by
blotting) with activity within the lysate but the latter
can certainly be measured.
     Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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