[Arkcon]

Currently, I have to perform enzyme assays, and I could use a few pointers on how they can be done to get the best results.  I've been in chemistry labs for a while now, but achieving the best accuracy and precision, and satisfying my coworkers that I'm handling the process appropriately could use some streamlining.  So I'm soliciting pointers from people who perform this sort of thing often.

Briefly, there's a variety of enzyme samples whose activity I need to determine, and a standardized enzyme control sample, you keep these on ice.  After making dilutions, you add the labeled substrate, and begin a 15 minute warm water bath reaction, then remove the mixture and halt the reaction.  Sounds simple ... if there's one tube.  The critical part of this procedure is managing a dozen tubes, consistent timing means you add substrate, begin heating, begin timing, then timing each addition so that you can remove each tube and add stop buffer after its 15.00 minutes are up.  An added wrinkle is that you have to reload the substrate pipette at some point, which adds a longer delay, and I have to compensate for the stop buffer addition. 

As I practice, my technique improves.  But, the sooner I streamline my procedure, the sooner I can be productive. So if anyone has tips, I'd like to hear them.

[Yggdrasil]

When I perform reactions like this, I set a timer to count up (rather than count down), and start the timer after I begin the first reaction.  Then after some period of time (e.g. 15 s), I'll start the next reaction, and continue until all the reactions have been initiated.  Then, 15 min after the first reaction has started, I'll stop the reactions in the same order in 15 s intervals, so the first reaction will have run from 00:00 to 15:00, the second reaction will have run from 00:15 to 15:15, the third reaction will have run from 00:30 to 15:30, etc.

The really tricky thing is enzyme assays where you have a 5s timepoint.  I had to learn how to pipette ambidextrously to pull that off.

[Babcock_Hall]

I may not be following you entirely, but it sounds as if Yggdrasil is performing a staggered start method, which we do also.  When we do beta-galactosidase stop-time assays, we use a ten minute incubation period.  We start 19 assays 30 seconds apart, then we go back to the first tube and add stopping solution again in 30 second intervals.  Typically we use the finger vortex method to mix the test tubes.  Is there any chance a second person could help out by counting off the time?  I would also give a great deal of thought to how you can pre-incubate the reaction at the correct temperature.  Lack of temperature control can sometimes be detected in continuous assays (RA John, Figure 10 in Chapter 2 of the book Enzyme Assays, edited by Eisenthal and Danson).

For reactions that are performed in a cuvette, Bel-Art manufactures plastic rods with small feet (originally designed by W W Cleland), and the enzyme solution is placed into the small reservoir in the foot.  The stirrer is rapidly moved up and down inside the cuvette to initiate the reaction.  I have been preparing a word document primarily on stop-time assays that discusses controls and other practical points.  You are welcome to read through it, if we can find a way to put it into your hands.

[Arkcon]

Thanks guys, I had thought my performance would improve with a stopwatch timer counting forward.  They seem to prefer a timer counting down, and I just don't get working that way.  Perhaps,  all I need is a longer pause between tubes, at least to start with.  The experts there rely on five sec interval.  I struggle with ten, and maybe I would like more time.

[Babcock_Hall]

Five seconds seems unreasonably tight.  10-15 seconds seems better.

[Babcock_Hall]

Here is a document I wrote on the subject of stop-time assays, but some of the points are also important in continuous assays.  At some points it makes reference to other similar documents that I wrote on pipetting, for example.  I may try to publish it at some point in a journal devoted to methodology.  Feel free to offer feedback.