Trouble shooting enzyme activity assay

wallan at uoguelph.ca wallan at uoguelph.ca
Sun Apr 13 21:55:42 EST 2003


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


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