Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-23 Thread Mohammad Khan
Dear Thomas, I do all my experiments in a 384 well, non-binding plates. I have a salt concentration of 50 mM NaCl, with BSA. i will try the other suggestions as well. >From a couple of experiments, I could determine the Kd to be approx. 10 nM. Thanks for all the suggestions! On Fri, Jul 21,

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-23 Thread Mohammad Khan
Dear Didier and Opher, The difference in polarities can reach upto 60 units. I have tried concentrations between 1-5 nM DNA. Maybe I will give higher concentrations a shot. Thanks! On Fri, Jul 21, 2017 at 4:02 PM, Didier Spittler wrote: > Hello, > > What is your

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-23 Thread Mohammad Khan
Dear Julius, That is a good suggestion. I will definitely try this. Thanks! On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius wrote: > Dear Mohammad, > your buffer is key to success in biophysical measurements. While your > protein may be totally fine in standard buffer, the

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-23 Thread Smith Liu
, 2017 9:44 AM To:CCP4BB@JISCMAIL.AC.UK 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 yo

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Phoebe A. Rice
r...@uchicago.edu> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton [eilise.mil...@gmail.com] Sent: Friday, July 21, 2017 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement Comple

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Ruud Hovius
if one has multiple species , e.g. bound and free, with each a different relative brightness and anisotropy/polarisation, then the use ( and interpretation) of anisotropy is straight forward; just a sum weighted by molecular fraction and the relative brightness. For polarisation is far more

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Keller, Jacob
>Ideally you should convert polarization to anisotropy. Simple enough – but >some referees can get picky… What is the argument for anisotropy being better? JPK

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Toth, Eric
With this type of behavior, one suspicion is that you didn’t let the reaction come to equilibrium prior to taking the measurement and the variability between time of mixing and measuring between individual replicates is introducing extra variability. Take a concentration at which you know there

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Morgan Milton
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

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Corbin Black
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

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Thomas Edwards
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

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Didier Spittler
Hello, What is your difference between the maximum and minimum value ? Have you try to change the probe concentration? Best, Didier Le 21 juil. 2017 15:34, "Mohammad Khan" a écrit : Dear all, I am trying to measure the difference in polarization upon the binding of

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Opher Gileadi
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