[RASMB] AUC capabilities

Thu Jan 27 07:49:34 PST 2011

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...

Mattia

At 09:51 AM 1/27/2011 -0500, Titus M. Franzmann wrote:

>Hi,
>
>Isn t there a correlation between, rotational correlation time and 
>particle shape and size?
>
>
>
>R(t) = R0exp(-1/tr) where R(t) is the decay, R0 is the max initial 
>anisotropy and tr the rotational correlation time (motility)
>
>And
>
>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.
>
>In this case it would indeed not be radius of gyration but the hydrodynamic.
>
>Anyway, I am certainly not certain about it and maybe this approach is too 
>complicated.
>
>Titus
>
>
>
>From: Mattia Rocco [mailto:mattia.rocco at istge.it]
>Sent: Thursday, January 27, 2011 9:10 AM
>To: Titus M. Franzmann; rasmb at server1.bbri.org
>Subject: Re: [RASMB] AUC capabilities
>
>
>
>
>Just a correction: from fluorescence anisotropy you don't get the radius 
>of gyration, but possibly the harmonic mean of the rotational correlation time.
>
>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...
>
>Mattia
>
>At 08:52 AM 1/27/2011 -0500, Titus M. Franzmann wrote:
>
>Ewa,
>Typically one tries to span a protein concentration range of at least 5times
>lower and 5times greater the kDa. Optimally 10times.
>So you would go at least from 2.5 to about 125 nM. Given the already low
>signal intensity of 0.1 at 220 at 25 nM you may not be able to observe the
>fully dissociated particle (monomers) and thus may not be able to acquired a
>dataset with a fully established monomer baseline. That would give an
>uncertainty in the Kd when fitting it to a mono-dimer association model
>(assuming no equilibrium intermediate populates).
>Anyway, I have carried out a couple of experiments at low signals, e.g. 0.05
>Abs. and determining the S-value is fairly robust, but fitting these low
>signal datasets to find proper Mw values is fairly difficult. Of course one
>can gain confidence by running triplicates and consolidate the datasets in a
>global fit. From this you can at least estimate what kind of particles you
>have. If the system is a fast interacting system, you should be able to use
>the Sw20avg values and plot them as the function of the protein
>concentration. It will be difficult to do this, when the system is slow
>interacting and the signal gets separated and distributed among the monomer
>and dimer fractions.
>Anyway, have you considered fluorescence anisotropy? According to your
>information, the protein contains several trp residues that you can use to
>monitor the assembly process. From the fluorescence anisotropy you can also
>gain information about the radius of gyration and make some estimate about
>the shape and size. Alternatively, we have recently used TEM to visualize
>monomers and dimers of a 39 kDa monomer. Surprisingly, we got some
>information using negative staining techniques and particle population
>analysis. You would at least get some idea about what you get.
>Titus
>
>Dr. Titus M. Franzmann
>S. G. Walter Lab
>Mol., Cell. and Develop. Biol. Dept.
>University of Michigan
>4140C Nat. Sci. Bldg
>830 N. University Ave.
>Ann Arbor, MI 48109-1048
>
>
>-----Original Message-----
>From: rasmb-bounces at server1.bbri.org 
>[<mailto:rasmb-bounces at server1.bbri.org>mailto:rasmb-bounces at server1.bbri.org]
>On Behalf Of Leech, AP
>Sent: Thursday, January 27, 2011 8:17 AM
>To: rasmb at server1.bbri.org
>Subject: Re: [RASMB] AUC capabilities
>
>Hi All,
>
>Eva, if you put all 50 ug into 120 uL for an equilibrium experiment,
>this would be ~0.4 mg/ml, or 7 uM, giving an approx absorbance of
>0.4 at 280 nm. If your Kd is right, this would practically all be
>dimer. To get down to sub-Kd concentrations, as Borries says, the
>absorbances would be impractically low.
>
>If you put it all into 400 uL for a velocity experiment, the initial
>concentration would be 0.125 mg/ml, 2.1 uM, absorbance ~0.13 at 280nm.
>This is well above the Kd also, I will leave someone with more
>experience to say if dissociation would affect the boundary detectably,
>I suspect not.
>
>It might be worth considering SPR. If you can attach one monomer to
>the chip surface without compromising dimerisation (practically I think
>you would attach the dimer and look for dissociation; there might be
>a problem with both halves of the dimer attaching), then wait for
>dissociation and flow various concentrations over the surface. The
>amounts are more in the ball park for what you have (say 10 ug for
>immobilisation and a few ug for each trial of free protein) though
>it's still not a lot! and you need to allow for control experiments
>for non-specific binding etc.
>
>Best regards,
>
>Andrew
>
>On 27/01/2011 04:29, Ewa Folta-Stogniew wrote:
> > At 10:39 PM 1/26/2011, you wrote:
> >> >
> >> > Hello,
> >> >
> >> > is it possible to determine dimerization constant in the order of 25
> >> > nM using AUC? What will be the minimal amount of protein needed?
> >> >
> >> > Ewa
> >> >
> >>
> >> Hi Ewa,
> >>
> >> the answer to your question is: it depends on several factors...
> >>
> >> 1. what optical system do you plan to use - there are choices:
> >> absorbance, Rayleigh interference, fluorescence emission
> >
> > absorbance or interference
> >
> >
> >> 2. If you use absorbance, there is a question about the extinction
> >> coefficient and the wavelength. Depending on extinction, 25 nM may
> >> be well within reach at several detection wavelengths.
> >
> > A280(0.1%)=0.85 (mg-1mL cm-1)
> >
> >> 3. Related to (2) is the question of size - the larger the protein,
> >> presumably the larger the extinction coefficient (more trp, tyr residues
> >> at 280 nm, more peptide backbone absorbance at lambda <= 230 nm). Ditto
> >> for Rayleigh interference - the larger the protein, the more refraction
> >> you will see and 25 nM could be well within range.
> >
> >
> > 59 kDa monomer
> >
> >
> >> 4. If you consider using the fluorescence detector, then 25 nM label
> >> of the right excitation/emission should be well within the range of
> >> detection.
> >
> > no fluorescence detection available
> >
> >
> >> 5. in order to get accurate Kds you would want to measure ideally
> >> at concentrations above and below the Kd to get good signal
> >> of the monomer and the dimer, so multiple concentrations measured
> >> either in velocity or equilibrium mode and globally fitted should
> >> give you the desired information. Related to that is that the
> >> Kd needs to be in the range where you are measuring. If your Kd is
> >> 100 micromolar and you are measuring at 25 nM and below, your answer
> >> is going to be quite unreliable. At the same token, measuring at
> >> 100 uM when your Kd is at 20 nM will not give you the desired result.
> >
> > Assuming Kd of 25 nM and the extinction coefficient as above- can one
> > determine Kd for dimerization from 50 micrograms of protein total (25
> > micrograms preferred)?
> >
> > If this is theoretically feasible, does anybody know of any published
> > AUC results for similarly low Kd?
> >
> > thanks,
> >
> > Ewa
> >
> >
> >> Your question about how much protein is needed at a minimum depends
> >> also on the questions I posed above. If you provide additional
> >> information we may be able to tell you more specifically for your sample.
> >>
> >> Regards, -borries
> >
> > _______________________________________________
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> > RASMB at rasmb.bbri.org
> > 
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>--
>Dr Andrew Leech                   *  Laboratory Head
>Technology Facility               *  Molecular Interactions Laboratory
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>University of York                *  Fax   : +44 (0)1904 328804
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