32P or 33P - Any difference in signal detection? (Dec/06/2004 )

Hi,

I am wanting to use 33P instead of 32P-ATP for a kinase reaction. Has anyone had experience with this and could tell me if there's any difference in the level of signal between these two? Thanks.

Hi elaineee...

I've nerver personnaly worked with 33P (only with 32P), but I think I still can help you. 33P is a lot "cleaner" as its emmision energy is lower. Maximum beta emission energy for 32P is 1.709 MeV and 0.249 MeV for 33P. So you'd have to use 7 times more 33P than 32P, wich means a whole lot more exposition to a film. Half life of 33P is longer though (25.4 day vs 14.3)... but it is so much more expensive...
I too perform kinase reaction (PKC) and use 32P, which has been perfect for me so far...
Hope this helped!!

Simon

I have one more consideration… As mention before, emission energy for 33P lower and very close to 35S, you will get much cleaner and sharpen bands. You need use single emulsion film (like Kodak BMR) and faced emulsion side to your gel. Using of “Amplify” from Amersham and -78 C can increase signal. But using of intesifyer screens which normally use for 32P, useless. I am personally, more like 33P because its low emission energy.

Alexei

Thanks
I normally dry my gel down and then read the signal from a phospherimaging screen with an imaging machine (Typhoon imager from Amersham). Would this allow the same detection as using film? Or is this the intensifying screen that isn't useful for 33P?
cheers.

I don't know if a phosphor-imager can read 33P. I'd say no, because of the low energy. Like Alexei said, 33P is pretty much like 35S. I'd rather expose the dried gel to a film, and scan it afterwards.
But there's no harm in trying exposing to the phosphor-imager... let me know how it worked...

Simon