In addition to everyone else's suggestions (especially trying greater [probe]), I would look at optimizing the following:
- Sufficient rxn volume. In the 384 well plates, I found that consistency between replicates improved with greater volume
- Read height
- Scaling
Corbin Black, BSc
MSc Biochemistry Candidate
University of Northern British Columbia
Department of Chemistry
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250-552-5046
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On Jul 21, 2017, at 7:11 AM, Thomas Edwards <[log in to unmask]<mailto:[log in to unmask]>> wrote:
A few tips, some/all of which may be relevant. Or not...
If you are in 96 or 384 well plates, they vary. We tried quite a few batches and brands before settling on one.
Keep your salt concentration as low as possible.
You should be able to measure Kd in a range of about 1nM to low uM. Outside that range is harder, or possibly impossible.
Buffers matter.
RNA binding proteins have preferred phosphates, PPIs Tris.
Some triton and/or some BSA can help.
All proteins are different, and each likes some or all or none of the above.....
Ideally you should convert polarization to anisotropy. Simple enough - but some referees can get picky...
Ed
T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT ?Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% evaporation, and 2% butterscotch ripple. ~Willy Wonka
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From: CCP4 bulletin board <[log in to unmask]<mailto:[log in to unmask]>> on behalf of Mohammad Khan <[log in to unmask]<mailto:[log in to unmask]>>
Reply-To: Mohammad Khan <[log in to unmask]<mailto:[log in to unmask]>>
Date: Friday, 21 July 2017 at 14:32
To: "[log in to unmask]<mailto:[log in to unmask]>" <[log in to unmask]<mailto:[log in to unmask]>>
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement
Dear all,
I am trying to measure the difference in polarization upon the binding of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in difference of polarization with decrease in protein concentration. However, the results are difficult to reproduce and also vary greatly within triplicates of an experiment.
Similar observations have been observed by my colleagues with their proteins.
Are there any tips or precautions to keep in mind while setting up these reactions?
Looking forward for suggestions.
Thank you.
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