Trouble shooting enzyme activity assay

EK anybody at uchicago.edu
Tue Apr 15 02:38:14 EST 2003


The volume of the buffer seem to have nothing to do with your problem. If
the assay worked with the crude extract and does not work with the clarified
extract, it means your protein goes into insoluble fraction. You should find
conditions allowing solubilization of the enzyme, and for that you can try
dozens of things that are all pretty well described in protein purification
method books. E.g., using 0.15-1.0M salt or low concentrations of detergents
(e.g. Tween-20 at 0.05%) in the extraction buffer might help sometimes. You
should really check plant protein purification books.
Emir


<wallan at uoguelph.ca> wrote in message
news:20030414025542.11273.qmail at ww02.hostica.com...
> I am trying to assay a novel enzyme in a plant extract. The enyzme
concentration is putatively low. I want to prepare a concentrated extract
from as little plant material as possible. The enzyme is reasonably stable.
The enzyme uses NADPH as a cofactor. I monitor the disappearance of the
absorbance of NADPH at 340 nm.
> The assay has worked with partially purified recombinant enzyme that was
expressed in bacteria, and has worked  with the plant crude extract.
>
> Questions:
>
>    What would be the minimum amount of extraction buffer I could use? 1
volume?
>  5 volumes??
>
>    I have already tried to extract 500 mg and 1 gm of tissue in 2x volume
buffer with little success. Would it be a good idea to extract large amounts
of tissue? And do I need to use a higher volume of extraction buffer and
then concentrate the extract later?  Or, could I use a minimum amount of
buffer to eliminate the concentration step later.
>
> Any comments would be helpful.
>
>
> WA
>
>
>
http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0





More information about the Proteins mailing list