发信人: moso (野鬼), 信区: Biology
标 题: Re: ATP binding assay
发信站: Unknown Space - 未名空间 (Thu Apr 14 13:00:50 2005), 转信

what do you mean no difference between control and samples? are they all blank
or all stained with isotope?
For whatever reason, it would be helpful to have a positive control in your
experiment if possible.
The incubation condition, I believe, is quite important for a successful
detection. The buffer component, like Megnesium,ADP concentration is somehow
critical. And also, the incubation time and temperature would make difference
sometime. So I would suggest that you might arrange an experiment to optimize
the incubation condition. It is easy, I think, to add more dots to the
membrane.

The washing condition is dependent on your primary results. If all blank, you
may need to reduce the washing strength. Otherwise, to increase the strength.

For detection, phosphoImager is not as sensitive as X-ray film, as far as I
know. So maybe it is a good idea to try X-ray film and expose at low
termperature for longer time if you got weak signal.

【 在 shagenxingbu (傻根行不) 的大作中提到: 】
: The protocol is as follows:
: 1. Incubate the sample at 37 degree for 10 min in the presence of
: alpha-P32-ATP(count: 500-1000 cpm)
: 2. Loading the sample onto membbranes:upper layer is Hybond, the down one is
: Nitrocellulose.
: 3. Vaccuming the sample.
: 4. Wash twice with buffer
: 5. Dry at RT
: 6. PhosphoImager
: The result: There is no difference between control and samples.
: Thanks
: 【 在 moso (野鬼) 的大作中提到: 】
: : There are several ways to detect the ATP binding. If you want
: trouble-shooting,
: : why not show us your original protocol?


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