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Enzyme Activity - efficiency of inhibition (Jan/05/2008 )

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QUOTE (mdfenko @ Jan 9 2008, 04:43 PM)
QUOTE (moljul @ Jan 9 2008, 05:08 AM)
another problem takes place during measurement of substrate cleavage: when i measure the same plate twice i get differences in my values. could this be due to temperature changes? could i somehow quench reaction to get reproduceable values? in some publications they quench in others they don´t.

reactions tend to continue unless stopped (or quenched). you may want to use one of the methods you found in the literature.


but how should i measure accurately, when values change from one to the other minute?

-moljul-

QUOTE (moljul @ Jan 10 2008, 09:24 AM)
but how should i measure accurately, when values change from one to the other minute?

you can:

1) read quickly, once.

or

2) stop the reaction then read at your convenience.

or

3) time the start of each reaction and read with the same intervals that you started the reactions.

-mdfenko-

QUOTE (mdfenko @ Jan 14 2008, 10:38 PM)
QUOTE (moljul @ Jan 10 2008, 09:24 AM)
but how should i measure accurately, when values change from one to the other minute?

you can:

1) read quickly, once.

or

2) stop the reaction then read at your convenience.

or

3) time the start of each reaction and read with the same intervals that you started the reactions.


i think i found out what my problem was. i measured too fast. my plate reader has no 37°C heating function. therefore it would be better to wait until the samples reach room temp. otherwise values could be falsified due to vapor. am i right?

-moljul-

QUOTE (moljul @ Jan 15 2008, 07:20 AM)
i think i found out what my problem was. i measured too fast. my plate reader has no 37°C heating function. therefore it would be better to wait until the samples reach room temp. otherwise values could be falsified due to vapor. am i right?

if they foul the optics.

do you stop the reaction? how?

is the substrate labile under the conditions? as an example, atp will continue to decompose in the conditions of the phosphate determination when performing an atpase assay.

-mdfenko-

QUOTE (mdfenko @ Jan 15 2008, 06:16 PM)
QUOTE (moljul @ Jan 15 2008, 07:20 AM)
i think i found out what my problem was. i measured too fast. my plate reader has no 37°C heating function. therefore it would be better to wait until the samples reach room temp. otherwise values could be falsified due to vapor. am i right?

if they foul the optics.

do you stop the reaction? how?

is the substrate labile under the conditions? as an example, atp will continue to decompose in the conditions of the phosphate determination when performing an atpase assay.



no, i don´t stopped the reaction. let stand for 5 min. values are stable.
substrate stability ok. i think. measure always assay buffer with substrate, and no signal is detected.

-moljul-

QUOTE (mdfenko @ Jan 7 2008, 07:56 PM)
as long as inhibitor is present inhibition will continue. if you remove inhibitor in later stages then inhibition will end and enzyme will become fully active.

in other words, reversible inhibitors don't bind to the enzyme permanently.



one question in context with this reply occurs: when enzymes are always inhibited in presence of an inhibitor, it could be also possible that the inhibitor (i applied in vivo) could be diluted and washed out during washing steps and lysis?
do i have to take care of this?

-moljul-

QUOTE (moljul @ Jan 15 2008, 02:49 PM)
QUOTE (mdfenko @ Jan 7 2008, 07:56 PM)
as long as inhibitor is present inhibition will continue. if you remove inhibitor in later stages then inhibition will end and enzyme will become fully active.

in other words, reversible inhibitors don't bind to the enzyme permanently.



one question in context with this reply occurs: when enzymes are always inhibited in presence of an inhibitor, it could be also possible that the inhibitor (i applied in vivo) could be diluted and washed out during washing steps and lysis?
do i have to take care of this?

yes. if the inhibitor is washed away or diluted to a less effective concentration then you will need to replenish it in order to maintain the inhibition (assuming that you want to continue inhibiting the enzyme).

-mdfenko-

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