Why is my restriction enzyme still working?

[BioTechie]

As a control in an experiment, I stored a sample ofrestriction enzyme to be used (3µL of Hind III) in a 37ºC incubator for 5 days- and it still worked just as well in a Lambda digest as samples stored at-20ºC! I wanted to show how the enzyme was inactivated, or at least lessactive, after storage at 37ºC - but couldn't. Why do all digest protocols recommend keeping these enzymes on ice while using them - when 5 days atat 37C didn't have an effect. Any ideas? I performed the test twice, not a pipet error because another control (no HindIII) showed no digestion as expected. I realize heat inactivation is 65C, but expected less activity at least.

 

Can't find an answer on the internet so far. Would like to find an enzyme that is less effective after being kept at that temperature.

 

Thanks.

 

*labelled gel attached

 

[Essay]

This is just guess, but it may not be the temperature of 37C specifically that the "warning" refers to as problematic, but rather the likelyhood of biological contamination, and subsequent growth --and consumption or biodegradation of the protein-- that the product warning is designed to prevent. You seem to have kept your samples uncontaminated.

 

You could test and see if the activity decreased for various contaminated samples?

 

~Good Luck

[CharonY]

The main factors are generally contamination with proteases, that destroy the enzyme. However, to me the restriction does not appear complete to me. Is it possible that the enzyme already lost a significant amount of activity? Also, what is the difference between the lanes labeled Lambda-HindIII and HindIII(temp), the bands appear to be identical.

 

For the test I would first aim to get a complete digestion taking the minimum required time, and use the RT-stored enzyme for the same amount. Even if it has reduced activity, if you treat it long enough you may still get significant digestion.

In the end the RT-storage will only result in a gradual reduction and you will have to time your experiments accordingly (but again, first go for complete digestion).