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 P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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