<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Hi, Christine,<div><br></div><div>Yes, that was a typo. Thanks for the correction.</div><div><br></div><div>We have been using the vbarD from le Maire (2000), rounding to 1.13.</div><div><br></div><div>Yes, our s and Mb are lower than one would expect for this protein if there were no detergent around.</div><div><br></div><div>Doing a very careful c(s), we find that there is a dominant, sharp peak, and well as a few minority peaks (~2-3% of sedimenting material) well separated from the main peak. I am therefore modeling all of that. The Mp that we get, after transforming with the (correct) formula, gives a number that agrees pretty well with SEC-MALS measurements on the same protein/detergent system. We have performed the SV experiment at several different concentrations, and all seem to give the same s, so we think that this is not a reaction boundary.</div><div><br></div><div>Thanks for your input!</div><div><br></div><div>Chad</div><div><br></div><div><br><div><div>On May 18, 2010, at 10:22 AM, Christine EBEL wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"> <div bgcolor="#ffffff" text="#000000"> <pre wrap="">Dear Chad,
Perhaps it was a typographic error, but Chad wrote:
" Mp = Mb (1-phiprime*rho),
where (neglecting bound lipid)
(1 - phiprime*rho) = (1 - vbarp*rho) + deltasubD(1-vbarD*rho)"
The first formula is inexact: The appropriate one is:
Mb = Mp(1-phiprime*rho)
</pre> ref for vbar LDAO=1.128-1.134(le maire) or 1.0597 (calculated, Maslennikov 2007)<br> ref: le Maire et al. Biochimica et Biophysica Acta 1508 (2000) 86-111<br> ref: Maslennikov et al BMC Structural Biology 2007, 7:74<br> So that (1 - phiprime*rho) < (1 - vbarp*rho) <br> And Mb and s are expected to be lower than for a soluble protein of same molar mass and frictional ratio. <br> The determination of Mb from one non -interacting particle analysis may be imprecise related to protein heterogeneity equilibrium of association-dissociation or unconsidered LDAO flotation (but you know...).<br> <br> All the best<br> Chritsine<br> <br> <br> <br> <br> <br> Le 17/05/2010 16:28, Mark Agacan a écrit : <blockquote cite="mid:4BF160B70200002400051D14@ia-gw-6.dundee.ac.uk" type="cite"> <pre wrap="">Hello,
I'm also running SV on a membrane protein in 150 mM NaCl, 50 mM NaCl,
and 0.1 % Foscholine-12.
I have no data for the detergent density or viscosity and so I tried to
omit it completely from the input parameters and see what sedfit c(s)
would output.
The mass values were way off from what was expected... as expected.
Can anyone suggest a way I can deal with density, viscosity, vbar
(without actually measuring the density) for this buffer / detergent
combination?
Cheers
Mark
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dr Mark Agacan, Scientific Officer for the Division of Biological
Chemistry and Drug Discovery,
Wellcome Trust Biocentre, College of Life Sciences, University of
Dundee, Dundee, DD1 5EH
Tel: +44 1382 386095 Fax: +44 1382 345764 Mobile: 07525 451 117
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
************************************************************
Please consider the environment. Do you really need to print this
email?
</pre> <blockquote type="cite"> <blockquote type="cite"> <blockquote type="cite"> <pre wrap="">Chad Brautigam <a class="moz-txt-link-rfc2396E" href="mailto:chad.brautigam@utsouthwestern.edu"><chad.brautigam@utsouthwestern.edu></a> 5/13/2010 15:41
</pre> </blockquote> </blockquote> </blockquote> <pre wrap="">Greetings, All, from the newbiest of membrane-protein newbies,
First, let me thank all of you experts who answered (both on- and off-
board) my last question on the topic of membrane proteins. 'Preciate
ya, as we say here in Texas.
I recently did some SV experiments on a membrane protein solubilized
in the accursed floating detergent LDAO (DDAO to some of you). I was
able to fit the data nicely in SEDFIT using a discrete species model.
By using the trick of setting the vbar to 0, I fitted for s and for
the buoyant molar mass (Mb). Hooray.
In a separate experiment, I used a combination of interference optics
and absorbance optics to measure the amount of detergent bound to the
protein. This was very consistent from sample to sample (I tried 2
different protein concentrations). This method is à la le Maire et al
(2008) Nat. Protocols, vol. 3, 1782.
OK, so I have Mb and I know the amount of detergent bound to the
protein on a g/g basis (this quantity is deltasubD).
We have the justly famous formula:
Mp = Mb (1-phiprime*rho),
where (neglecting bound lipid)
(1 - phiprime*rho) = (1 - vbarp*rho) + deltasubD(1-vbarD*rho)
So, since I have Mb and deltasubD, and I know rho and the vbars, can I
just plug the Mb that I get from SV into these equations and get a
good estimate for Mp, or is there some SV subtlety here that my newbie
brain hasn't apprehended? I know this can be done for SE, but I've
never seen anyone attempt it for SV.
Any and all responses will be 'preciated.
Chad
======================================
Chad A. Brautigam, Ph.D.
Assistant Professor
Department of Biochemistry
The University of Texas
Southwestern Medical Center at Dallas
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
Office: 214-645-6384
Fax: 214-645-6353
Email: <a class="moz-txt-link-abbreviated" href="mailto:chad.brautigam@utsouthwestern.edu">chad.brautigam@utsouthwestern.edu</a>
_______________________________________________
RASMB mailing list
<a class="moz-txt-link-abbreviated" href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a>
<a class="moz-txt-link-freetext" href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a>
The University of Dundee is a registered Scottish charity, No: SC015096
</pre> <pre wrap=""><fieldset class="mimeAttachmentHeader"></fieldset>
_______________________________________________
RASMB mailing list
<a class="moz-txt-link-abbreviated" href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a>
<a class="moz-txt-link-freetext" href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a>
</pre> </blockquote> <br> <br> <pre class="moz-signature" cols="72">--
Christine EBEL
Institut de Biologie Structurale CEA-CNRS-UJF
41 rue Jules Horowitz, F-38027 Grenoble France
Tel (33) (0) 4 38 78 95 70; Fax (33) (0) 4 38 78 54 94
<a class="moz-txt-link-abbreviated" href="mailto:christine.ebel@ibs.fr">christine.ebel@ibs.fr</a>
<a class="moz-txt-link-freetext" href="http://www.ibs.fr/laboratories/molecular-biophysics-lab-lbm/ssimpas-435/">http://www.ibs.fr/laboratories/molecular-biophysics-lab-lbm/ssimpas-435/</a>
</pre> </div> </blockquote></div><br></div></body></html>