chemiluminescence imaging and quantitation

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Sat Feb 10 05:47:09 EST 2001


In article
<FE6A25586CA68D4DB3A7295E737FAEB507E3C3 at neuronet.neuro.neurovir.com>,
  kdolter at neurovir.com (Karen Dolter) wrote:
> What is a good system for imaging and quantitation of nonradioactive
blots?
> Has anyone tried Syngene's GeneGnome?

What I like to do is dot blots, using the BioDot (BioRad) vacuum
manifold and Immobilon membranes. Each well is filled with 100 ul
buffer, then the sample is added and the vaccum put on. Once the sample
has been sucked through the membrane, the blot is developed with primary
 and HRP-coupled secondary antibody like a Western. Then the blot is
incubated in ECL-reagent (Amersham, but similar reagents are available
from other manufacturers, or use home made) for 10 min, then placed into
a holder which is inserted into a 96-well chemoluminescence reader
(Berthold, Packard, ...). You do not need expensive flash-kinetic
equipment for this, simple glow kinetics will do.

This assay gives linear signal vs [Antigen] plots over several orders of
magnitude, and is very sensitive as essentially all the antigen in the
sample is bound to the membrane, rather than a few % as in an ELISA. I
used it once to follow the purification of a protein, for which we had
only an antibody, but no functional assay.

Of course you have to be sure that your primary antibody is specific,
i.e. reacts only with one protein in your sample (Western-blott!). Also,
you need to mark two corner holes on the membrane before the
filtration, so that you can later align the spots with the holes
in the holder.


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