enzyme assay - (Feb/16/2007 )

hey
please leave your suggestions on cautions of enzyme assay
like how to maintain timing, about using pippetteman, reading the sample etc etc

sometimes i cannot maintain exact time, like i become late for 2-3 seceonds, is it a big deal???
how about using 5 ml pippetteman for adding 2-5 ml soln?? or glass pippetting is better
at the time of reading do u wash the glass cell everytime you change the sample and do u set auto zero everytime you change the sample
and so on.......................
thanks

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managing time is one of most important challenge in enzyme assaying; deviation of some seconds should be expressed as S.E.M., and can be integrated in a plot

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Have everything on ice while you pipette all the tubes and then place the tubes in 37C and start the countdown for the experiments. This way one ensures equal timing for all tubes. Time is important for enzymatic experiments.

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seems no one is interested to leave some suggestions about enzyme assay

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Here's a couple :-

ALWAYS make fresh stocks on the day of the assay. ESPECIALLY if you are adding co-factors.

In the Kinetic enzyme assays I have done, add the SUBSTRATE to initiate the reaction i.e Buffer first, enyme second, followed by cofactors THEN sunbstrate at time 0

DO NOT BE OVER AMBITIOUS...do a proof of principle experiment FIRST in a SINGLE CUVETTE.

ALWAYS OPTIMISE ALL THE CONDITIONS because obtaining a protocol from the literature is fraught with danger i.e. in a materials/method section there is more information left OUT that left in.

How's that

Rhombus

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thanks a lot, waiting for more commentssssssss from others as well
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