May i ask, whether the fluoresnce anisotropy method was reliable enough to
determine the stoichiometry of a protein complex?
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在2017年07月22日 03:44，Phoebe A. Rice 写道：
You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the
well?
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Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
[email protected]
From: CCP4 bulletin board [[email protected]] on behalf of Morgan Milton
[[email protected]]
Sent: Friday, July 21, 2017 9:44 AM
To:[email protected]
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement
Completely agree, you need a higher DNA concentration. We have had luck with 10
nM DNA.
Also, bubbles have a HUGE impact on how the fluorescent signal is measured.
Make sure you spin your plates down (assuming you are using them) to remove any
bubbles. We just had an undergrad read his anisotropy assay and the data looked
horrible. He then realized he had not spun his plate down, after doing so the
data was much more consistent.
Are you doing technical replicates? We do at least triplicates per plate.
All the best,
Morgan
Morgan E. Milton, PhD
On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi <[email protected]>
wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence
signal is too low, you will still get a ratio but it will be essentially noise.
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in
the low nM range, you may have to use other methods to measure KD.
