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Non-radioactive DNA-PK activity assays?

dna-pk radioisotope radioactive non-radioactive

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6 replies to this topic

#1 JDSBlueDevl

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Posted 20 November 2014 - 03:18 PM

I'm trying to find a DNA-PK activity assay that does not depend on radiolabeling with P-32.  The kit that was written into my mentor's grant (and the one that almost everyone seems to use) is the Promega SignaTECT kit, which depends on radiolabeling with P-32.  Our laboratory is not certified to use radioisotopes (only certified to use the gamma irradiator), so this kit is of no use.  I have told my mentor repeatedly after exhaustive Google searches that there are no non-radioactive DNA-PK activity assay kits, but he insists on saying that there is (why he hasn't pointed the one he's thinking about to me, I don't know).  Maybe somebody here might know of the existence of one, I hope.

 

Note: I am not looking for the DNA-PK ELISA kit that measures DNA-PK expression.  I saw that and was excited until I saw that it is only good for protein expression, not enzymatic activity.



#2 bob1

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Posted 20 November 2014 - 03:38 PM

I suspect that part of the problem is that any molecules that have a large non-radioactive addition like DIG or similar would interfere with the PK processivity, hence the use of P32. It might be possible to adapt the kit to use a stable isotope labeling approach, but this would be dependent on the ability to detect the change via mass spec.



#3 JDSBlueDevl

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Posted 20 November 2014 - 07:21 PM

So, I guess that means I have to collaborate with a lab that is certified for radioisotopes? If that's the case, does that mean I can do the experiment under their supervision, or does it have to be farmed off to comply with an NRC regulation? At least I can back up the argument with my mentor that a nonradioactive assay is impossible.

#4 bob1

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Posted 21 November 2014 - 12:34 AM

My post is just a guess. The regulations depend on where you are, but I think in most western countries, you should be able to do the experiment under supervision of the accredited lab.



#5 mdfenko

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Posted 21 November 2014 - 04:48 AM

after being trained in the use and handling of radioactive materials.

 

in the us, more stringent local regulations supersede national regulations.


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#6 JDSBlueDevl

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Posted 22 November 2014 - 07:13 PM

I don't know whether DC is stricter than the federal regulations (I would imagine they would be identical).  I have already received the training and taken the courses, but as far as I know, I am only filling out NRC forms for using the gamma irradiator.  I would hope the forms also apply to radioisotopes, but that may be wishful thinking.



#7 mdfenko

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Posted 24 November 2014 - 06:01 AM

record keeping will be in the lab in which you perform the experiment. they should be able to show you what they need.

 

and dc could be different from the national requirements. nyc has more stringent requirements than national and nys.


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