I see my colleagues pending the arrival of the command they work with different outdated restriction enzymes in many reactions when I ask them to stop these reactions because we must respect the suppliers recommendations and there are enzymatic parameters to respect namely the Michaelis constant ..... they continue to conduct their experiments. Certainly they get results but I find that these results are uncertain, enzymes continue to function even outdated but I think that results are unreliable. Suppliers sometimes put an end to the production of certain buffers such as OPA (One Phor All buffer). Who can tell me which are the enzymatic parameters altered when enzymes are outdated and why some suppliers stop production of certain enzyme buffers? Thanks
I recently re-assayed some restrictions that had been stored at -20 for 15 - 20 years. A couple of duds, but the rest were as good as new.
The expiry date often just reflects the longest time the manufacturer has had the product in storage and checked it.
The manufacturers of restriction enzymes probably put expiration dates on their products to advise users of how long they can be sure the enzymes will retain their full activity, if stored correctly. After that date, they don't guarantee that the activity will not decrease. However, that does not mean the enzymes won't still be useful after the expiration date. The activity may show some decrease, but this can be compensated by using a higher concentration, up to a point. The added protein, glycerol in some cases, and additional sample dilution resulting from adding a larger volume of the enzyme may become a problem for sample purification, and the enzyme reaction may go slower.
Unreliable? More like inefficient I think. Expired enzymes will only cause the enzyme to be no longer satisfying the standard. No harm like misidentifying the restriction sites or anything like that. As time goes on, enzymes like any other protein will slowly denatured and lost its function, hence less active enzymes available and making it less efficient. So more enzymes/time required to cut the same amount of plasmid.
As for the buffer problem, is it possible to gain access to the buffer component list? If it is, then probably you could make your own or find some work around that has the similar properties to the buffer you needed.
Hope that help,
Radif
Update:
I just checked the Question I Followed and notice that there is another answer and found that there is a similar answer above mine, and it is even 3 hours previously. I remember it well that there is no answer at all before, and there is even "Be the first to answer" tag below the question.
Oh whatever, I just wanna add, the restriction enzyme is not going to be used for quantification purpose, so I think there won't be much trouble.
Hi, my own experience with out of date restriction enzyme showed that the results obtained are reliable! Depends how old are the RE but I have very good results with even 3-4 years old enzymes, but of course they were stored at the right conditions.
I recently re-assayed some restrictions that had been stored at -20 for 15 - 20 years. A couple of duds, but the rest were as good as new.
The expiry date often just reflects the longest time the manufacturer has had the product in storage and checked it.
thank you for your help. The question still remains with no response what enzymatic prameters that may be altered ? Can we use these enzymes in the long term 30 years 40 years ...?
If the enzyme is unstable and loses activity, you will most likely see a decrease in Vmax, but no change in Km. This is because Vmax depends on the concentration of enzyme active sites, but Km depends on the structure of the active sites, not the concentration of them. Because protein folding is highly cooperative, an individual enzyme molecule will probably exist in either the fully active or completely inactive state, not something in between.
Andrew Jenkins' answer shows that some restriction enzymes are extremely stable and others are not. If for some reason you wanted to keep them for decades, you should probably store them in liquid nitrogen. The cost of storing them for such a long time would probably exceed the cost of buying them, and it would also be inconvenient to use them.
I have no problems with using outdated restriction enzymes as you will immediately see on the gel if the have worked (i.e. produced the expected pattern of bands on an agarose gel) or if their activity is low (i.e. leave much of the input DNA undigested). Reliability is not an issue and I am not aware that storage increases the star activity. So for most applications they should be fine.
From personal experience, I can say that I have had some problem with outdated EcoRI, but this was immediately obvious from the gel (many more bands than expected and later verified using new EcoRI); if you are using EcoRI, make sure to validate its activity.
According to http://www.cmbn.no/tonjum/repuffer.html, One-Phor-All Buffer PLUS (OPA+) is 50 mM potassium acetate, 10 mM Tris-acetate, pH 7.5, 10 mM magnesium acetate. OPA is 100 mM Potassium glutamate, 25 mM Tris-acetate, pH 7.5, 10 mM Magnesium acetate, 50 µg/ml Bovine serum albumin, 0,5 mM 2-mercaptoethanol.
I have a fundamental problem with all these kits out there whose composition is kept secret. How is one to reproduce experiments when the manufacturer stops making it? This corrupts the very core of science. Kits are ok, but only if you can do the experiment without them first.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes