Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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, 2017 at 4:11 PM, Thomas Edwards <t.a.edwa...@leeds.ac.uk>
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
>
>
>
> *From: *CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Mohammad
> 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
>
>
>
> 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.
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 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
> 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.
>
>
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 large and hydrophobic
> fluorophores used in FP, MST and other assays are prone to adsorbing to
> plasticware etc. Also, at 1nM concentration, you are losing much more of
> your protein to adsorption, so you will have far less active protein at
> those concentrations than calculated. I would try to add detergent and, if
> necessary, BSA to your buffer. For my assay development projects, Tween20
> or Brij35 (0.03%) have worked well and I have used 0.5% BSA. Once you get
> the buffer right, measurements will be far more reproducible. Also, try to
> include a good control, e.g. DNA with Cy3, but sequence that does not bind
> to test for nonspecific, concentration dependent effects.
> I hope this helps, if you have any questions, feel free to email me!
> Best,
> Julius
>
>
> > On 21 Jul 2017, at 15:32, Mohammad Khan  wrote:
> >
> > 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.
>
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Smith Liu]

May i ask, whether the fluoresnce anisotropy method was reliable enough to 
determine the stoichiometry of a protein complex?


发自网易邮箱大师


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?




++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@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


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 <opher.gile...@sgc.ox.ac.uk> 
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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Phoebe A. Rice]

You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu<mailto:pr...@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

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 
<opher.gile...@sgc.ox.ac.uk<mailto:opher.gile...@sgc.ox.ac.uk>> 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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 complicated.

See e.g. Lakowicz - Principles of fluorescence spectroscopy.

However, as long as one treats & interprets the data correctly there is 
no principle reason to forbid the use of polarisation.


Personally, I prefer anisotropy.

Ruud


On 21/7/17 17:57, Keller, Jacob wrote:


*>*Ideally you should convert polarization to anisotropy. Simple 
enough – but some referees can get picky…


What is the argument for anisotropy being better?


JPK



--

Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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

[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 is a significant 
signal change and measure the polarization every few minutes for an hour. You 
might find that you need to pre-incubate the samples for an extended time prior 
to taking measurements.

With that said, my first suspicion is that you have lousy binding.

Eric


From: CCP4 bulletin 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. 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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 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 
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.
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 Biochemistry Candidate
University of Northern British Columbia
Department of Chemistry
bla...@unbc.ca<mailto:bla...@unbc.ca>
blackcor...@gmail.com<mailto:blackcor...@gmail.com>
250-552-5046

Sent from my iPhone

On Jul 21, 2017, at 7:11 AM, Thomas Edwards 
<t.a.edwa...@leeds.ac.uk<mailto:t.a.edwa...@leeds.ac.uk>> 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


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Mohammad Khan 
<mohdkhan0...@gmail.com<mailto:mohdkhan0...@gmail.com>>
Reply-To: Mohammad Khan <mohdkhan0...@gmail.com<mailto:mohdkhan0...@gmail.com>>
Date: Friday, 21 July 2017 at 14:32
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
<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 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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 
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
[cid:image001.png@01D30233.9026B5C0]

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Mohammad 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

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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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
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.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[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 measure KD.

[ccp4bb] Off topic: Flourescence anisotropy measurement

[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 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.