Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-23 Thread Mohammad Khan
t; Khan <mohdkhan0...@gmail.com> > *Reply-To: *Mohammad Khan <mohdkhan0...@gmail.com> > *Date: *Friday, 21 July 2017 at 14:32 > *To: *"CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> > *Subject: *[ccp4bb] Off topic: Flourescence anisotropy measurement >

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
board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad Khan Sent: Friday, July 21, 2017 9:33 AM To: CCP4BB@JISCMAIL.AC.UK 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

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
<CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 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 varyin

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Thomas Edwards
gmail.com> Reply-To: Mohammad Khan <mohdkhan0...@gmail.com> Date: Friday, 21 July 2017 at 14:32 To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement Dear all, I am trying to measure the difference in polariz

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

[ccp4bb] Off topic: Flourescence anisotropy measurement

2017-07-21 Thread Mohammad Khan
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