Chemicon protein concentrate kit - what's in it?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Fri Aug 27 10:47:12 EST 1999


In article <RNA.world-2708991716260001 at expmac19.uni-muenster.de>,
Juergen Brosius <RNA.world at uni-muenster.de> writes
> Concentration needs to be done between purification
>steps (i.e. we cannot run the material afterwards on an SDS gel yet and
>expect a few protein bands).  The mixture is still complex and we need to
>go on to the next purification step without risking to blow the complex
>apart.  Frank mentioned Ammonium sulfate precipitation and PEG.  After the
>former we had trouble resuspending and PEG did not precipitate
>quantitatively.  Any suggestions concerning protocol variations?  We
>thought of testing concentration units such as Amicon.

Have you tried vacuum dialysis?

Take the really narrow bore tubing (1/4 inch?), tie one off end securely
and place other end through a hole in a bung so it comes through the
other side. 

Fill with your RNP solution and use a funnel (we used to use glass
chromatography columns drawn out at one end) to hold the tubing in
place.

Put the whole thing onto a side arm flask filled with dialysis buffer
and stirrer bar.

Attach water vacuum pump (not too much vacuum) and pray.

It will concentrate 100ml down to a ml or so quite quickly. However make
sure the knot at the end is intact and don't use dialysis tubing greater
then 1/4 inch. As the vacuum goes on the tubing will expand in size.
With wider tubing it will burst!

Another alternative is much safer. Take sample into dialysis tubing, tie
off at both ends and place in dry PEG8000 or surround with dry sucrose
or sephadex. 

We use this with PEG to concentrate (final step prior to dialysis
against a 50% glycerol storage buffer) virtually every enzyme we produce
and haven't lost any in over ten years. The only problem is that you can
concentrate to dryness if you do not keep a close eye. You can
concentrate litres with enough PEG etc. PEG stays on outside and will
not pass through the membrane but obviously sucrose will.

Duncan   
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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