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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Hi, <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Isn’t there a correlation between, rotational correlation time and particle shape and size?<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>R(t) = R0exp(-1/tr) where R(t) is the decay, R0 is the max initial anisotropy and tr the rotational correlation time (motility)<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>And<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>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.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>In this case it would indeed not be radius of gyration but the hydrodynamic.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Anyway, I am certainly not certain about it and maybe this approach is too complicated. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Titus<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> Mattia Rocco [mailto:mattia.rocco@istge.it] <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<o:p></o:p></span></p></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><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><o:p></o:p></p><p class=MsoNormal>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>> _______________________________________________<br>> RASMB mailing list<br>> RASMB@rasmb.bbri.org<br>> <a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><br><br>-- <br>Dr Andrew Leech * Laboratory Head<br>Technology Facility * Molecular Interactions Laboratory<br>Department of Biology (Area 15) * Tel : +44 (0)1904 328723<br>University of York * Fax : +44 (0)1904 328804<br>PO Box 373, York YO10 5YW * Email : apl3@york.ac.uk<br>EMAIL DISCLAIMER: <a href="http://www.york.ac.uk/docs/disclaimer/email.htm">http://www.york.ac.uk/docs/disclaimer/email.htm</a><br>_______________________________________________<br>RASMB mailing list<br>RASMB@rasmb.bbri.org<br><a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><br><br><br>_______________________________________________<br>RASMB mailing list<br>RASMB@rasmb.bbri.org<br><a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><o:p></o:p></p><p><span style='font-size:10.0pt;font-family:"Arial","sans-serif"'><br></span><span style='font-size:10.0pt;font-family:"Verdana","sans-serif"'>ATTENZIONE.<br>Il presente messaggio ed i suoi allegati devono intendersi ad uso esclusivo dei suoi destinatari e sono confidenziali. 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