I am trying to find the optimum amount of M-Mlv rt enzyme (purified by myself) required for a RT-PCR . I compared the activity of my M-Mlv rt enzyme and other commercially available rt enzyme.I found that my enzyme have same activity as commercial one when it dilute to 10 fold.Then I run SDS-PAGE (same amount of protein) and got to know that my rt enzyme has very low amount of protein compare to commercial rt enzyme but it had the same activity.After then, I diluted the commercial rt enzyme and checked its' activity. Surprisingly, it had the same activity even I diluted it for 40 time.Major question is why they(commercial ) are adding more amount of protein even small ( nearly 40 times less) amount of rt enzyme can produce same activity.
There are a few obvious reasons:
1. Most people never use just the right amount of enzyme that is needed for the task.
2. Not everyone is careful enough to have no contaminants that interfere with enzyme activity.
3. Less than ideal conditions of handling and storage causes partial loss of activity. Excess ensures that the results are still not completely disappointing.
etcetera...
Dear
In my opinion, you need at first time to measure activity of enzyme and at second time to verify the degree of purity of your isolated enzyme.
Cordialy
Marius
Thank you all for your answers.
@ Aqleem Abbas- I purified the enzyme using Ni-Nta column since it is a his tag protein.
@ Tausif Alam-commercial rt enzyme had a single band in SDS- PAGE. If it is due to the contaminants why they ask us to add more units? More units of protein has more contaminants.Therefore, by adding more units it should not give any activity.But it had its' activity even we add 200 units. The question is why they ask us to add more units even less no of units has same activity.
@ Marius K. Somda-As you mentioned, I checked the activity and found that more amount of enzyme no activity.But when I diluted, it had the activity.It may be due to the contaminants remain even after the purification.
I believe that you misunderstood my point about contaminants, which was to do with a user's sample, not the commercially supplied enzymes. All major vendors have been in business for decades and it is not unreasonable to assume that their purification methods are quite well established for quality and consistency.
Premise of such reactions as you are discussing, is that the limiting amount in your reaction is the key substrate, in your case, the RNA - and not the enzyme.
"Standard Conditions" are usually described to give you considerable latitude on the side of key substrate, such that if you have more of it in some reactions, you do not run out of the enzyme and dNTPs etc.
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