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<font size=3><br>
Sure, you are correct, what you can get is the hydrodynamic volume, hence
a measure of the rotational hydrodynamic radius. I just wanted to point
out that it wasn't the radius of gyration...<br><br>
Mattia<br><br>
At 09:51 AM 1/27/2011 -0500, Titus M. Franzmann wrote:<br><br>
<blockquote type=cite class=cite cite>Hi, <br><br>
Isn t there a correlation between, rotational correlation time and
particle shape and size?<br><br>
<br><br>
R(t) = R0exp(-1/tr) where R(t) is the decay, R0 is the max initial
anisotropy and tr the rotational correlation time (motility)<br><br>
And<br><br>
Tr=nV/kT (Stokes-Einstein), where n is the viscosity, V is the
hydrodynamic volume for a spherical particle and kT is the Bolzmann
constant and Temperature.<br><br>
In this case it would indeed not be radius of gyration but the
hydrodynamic.<br><br>
Anyway, I am certainly not certain about it and maybe this approach is
too complicated. <br><br>
Titus<br><br>
<br><br>
<b>From:</b> Mattia Rocco
[<a href="mailto:mattia.rocco@istge.it" eudora="autourl">mailto:mattia.rocco@istge.it</a>]
<br>
<b>Sent:</b> Thursday, January 27, 2011 9:10 AM<br>
<b>To:</b> Titus M. Franzmann; rasmb@server1.bbri.org<br>
<b>Subject:</b> Re: [RASMB] AUC capabilities<br><br>
<br><br>
<br>
Just a correction: from fluorescence anisotropy you don't get the radius
of gyration, but possibly the harmonic mean of the rotational correlation
time.<br><br>
As for using TEM with negative staining, one has to be absolutely sure
that adhesion on the grid doesn't influence the monomer-dimer
equilibrium...<br><br>
Mattia<br><br>
At 08:52 AM 1/27/2011 -0500, Titus M. Franzmann wrote:<br><br>
Ewa,<br>
Typically one tries to span a protein concentration range of at least
5times<br>
lower and 5times greater the kDa. Optimally 10times.<br>
So you would go at least from 2.5 to about 125 nM. Given the already
low<br>
signal intensity of 0.1 at 220 at 25 nM you may not be able to observe
the<br>
fully dissociated particle (monomers) and thus may not be able to
acquired a<br>
dataset with a fully established monomer baseline. That would give
an<br>
uncertainty in the Kd when fitting it to a mono-dimer association
model<br>
(assuming no equilibrium intermediate populates).<br>
Anyway, I have carried out a couple of experiments at low signals, e.g.
0.05<br>
Abs. and determining the S-value is fairly robust, but fitting these
low<br>
signal datasets to find proper Mw values is fairly difficult. Of course
one<br>
can gain confidence by running triplicates and consolidate the datasets
in a<br>
global fit. From this you can at least estimate what kind of particles
you<br>
have. If the system is a fast interacting system, you should be able to
use<br>
the Sw20avg values and plot them as the function of the protein<br>
concentration. It will be difficult to do this, when the system is
slow<br>
interacting and the signal gets separated and distributed among the
monomer<br>
and dimer fractions.<br>
Anyway, have you considered fluorescence anisotropy? According to
your<br>
information, the protein contains several trp residues that you can use
to<br>
monitor the assembly process. From the fluorescence anisotropy you can
also<br>
gain information about the radius of gyration and make some estimate
about<br>
the shape and size. Alternatively, we have recently used TEM to
visualize<br>
monomers and dimers of a 39 kDa monomer. Surprisingly, we got some<br>
information using negative staining techniques and particle
population<br>
analysis. You would at least get some idea about what you get.<br>
Titus<br><br>
Dr. Titus M. Franzmann<br>
S. G. Walter Lab<br>
Mol., Cell. and Develop. Biol. Dept. <br>
University of Michigan<br>
4140C Nat. Sci. Bldg<br>
830 N. University Ave.<br>
Ann Arbor, MI 48109-1048<br><br>
<br>
-----Original Message-----<br>
From: rasmb-bounces@server1.bbri.org
[<a href="mailto:rasmb-bounces@server1.bbri.org">mailto:rasmb-bounces@server1.bbri.org</a>]<br>
On Behalf Of Leech, AP<br>
Sent: Thursday, January 27, 2011 8:17 AM<br>
To: rasmb@server1.bbri.org<br>
Subject: Re: [RASMB] AUC capabilities<br><br>
Hi All,<br><br>
Eva, if you put all 50 ug into 120 uL for an equilibrium
experiment,<br>
this would be ~0.4 mg/ml, or 7 uM, giving an approx absorbance of<br>
0.4 at 280 nm. If your Kd is right, this would practically all be<br>
dimer. To get down to sub-Kd concentrations, as Borries says, the<br>
absorbances would be impractically low.<br><br>
If you put it all into 400 uL for a velocity experiment, the
initial<br>
concentration would be 0.125 mg/ml, 2.1 uM, absorbance ~0.13 at
280nm.<br>
This is well above the Kd also, I will leave someone with more<br>
experience to say if dissociation would affect the boundary
detectably,<br>
I suspect not.<br><br>
It might be worth considering SPR. If you can attach one monomer to<br>
the chip surface without compromising dimerisation (practically I
think<br>
you would attach the dimer and look for dissociation; there might
be<br>
a problem with both halves of the dimer attaching), then wait for<br>
dissociation and flow various concentrations over the surface. The<br>
amounts are more in the ball park for what you have (say 10 ug for<br>
immobilisation and a few ug for each trial of free protein) though<br>
it's still not a lot! and you need to allow for control experiments<br>
for non-specific binding etc.<br><br>
Best regards,<br><br>
Andrew<br><br>
On 27/01/2011 04:29, Ewa Folta-Stogniew wrote:<br>
> At 10:39 PM 1/26/2011, you wrote:<br>
>> ><br>
>> > Hello,<br>
>> ><br>
>> > is it possible to determine dimerization constant in the
order of 25<br>
>> > nM using AUC? What will be the minimal amount of protein
needed?<br>
>> ><br>
>> > Ewa<br>
>> ><br>
>><br>
>> Hi Ewa,<br>
>><br>
>> the answer to your question is: it depends on several
factors...<br>
>><br>
>> 1. what optical system do you plan to use - there are
choices:<br>
>> absorbance, Rayleigh interference, fluorescence emission<br>
><br>
> absorbance or interference<br>
><br>
><br>
>> 2. If you use absorbance, there is a question about the
extinction<br>
>> coefficient and the wavelength. Depending on extinction, 25 nM
may<br>
>> be well within reach at several detection wavelengths.<br>
><br>
> A280(0.1%)=0.85 (mg-1mL cm-1)<br>
><br>
>> 3. Related to (2) is the question of size - the larger the
protein,<br>
>> presumably the larger the extinction coefficient (more trp, tyr
residues<br>
>> at 280 nm, more peptide backbone absorbance at lambda <= 230
nm). Ditto<br>
>> for Rayleigh interference - the larger the protein, the more
refraction<br>
>> you will see and 25 nM could be well within range.<br>
><br>
><br>
> 59 kDa monomer<br>
><br>
><br>
>> 4. If you consider using the fluorescence detector, then 25 nM
label<br>
>> of the right excitation/emission should be well within the range
of<br>
>> detection.<br>
><br>
> no fluorescence detection available<br>
><br>
><br>
>> 5. in order to get accurate Kds you would want to measure
ideally<br>
>> at concentrations above and below the Kd to get good
signal<br>
>> of the monomer and the dimer, so multiple concentrations
measured<br>
>> either in velocity or equilibrium mode and globally fitted
should<br>
>> give you the desired information. Related to that is that
the<br>
>> Kd needs to be in the range where you are measuring. If your Kd
is<br>
>> 100 micromolar and you are measuring at 25 nM and below, your
answer<br>
>> is going to be quite unreliable. At the same token, measuring
at<br>
>> 100 uM when your Kd is at 20 nM will not give you the desired
result.<br>
><br>
> Assuming Kd of 25 nM and the extinction coefficient as above- can
one<br>
> determine Kd for dimerization from 50 micrograms of protein total
(25<br>
> micrograms preferred)?<br>
><br>
> If this is theoretically feasible, does anybody know of any
published<br>
> AUC results for similarly low Kd?<br>
><br>
> thanks,<br>
><br>
> Ewa<br>
><br>
><br>
>> Your question about how much protein is needed at a minimum
depends<br>
>> also on the questions I posed above. If you provide
additional<br>
>> information we may be able to tell you more specifically for
your sample.<br>
>><br>
>> Regards, -borries<br>
><br>
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