[RASMB] still Beckman software

Jo Butler pjgb at mrc-lmb.cam.ac.uk
Wed Oct 24 04:48:01 PDT 2001


John,

If I have given the impression that I think the fringe count is not 
wavelength dependent, this is clearly false.  What I was meaning to say is 
that if one uses the refractive increment for a specific wavelength, then 
there is probably no need to enter the wavelength as such into the 
calculation, since the increment is already determined by the wavelength.

Yours,

Jo

--On Tuesday, October 23, 2001 10:28 am -0700 John Philo 
<jphilo at mailway.com> wrote:

> The older archived RASMB emails can be found at:
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>
> and current archives at
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> http://rasmb-email.bbri.org/pipermail/rasmb/
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> -------------------------------------------------------------------------
> ---------
>
> Kristian,
>
> Sorry, I don't completely understand your question about that specific
> simulation, but yes I believe the simulations are correct. They certainly
> match quite closely what I see in real absorbance experiments, and that
> simulator is very handy for planning equilibrium experiments.
>
> You can ignore the whole units and conversion factors issue by simply
> entering your loading concentrations in instrument units (fringes or
> absorbance) and leaving the conversion factor at 1. If you do that the
> simulated curves will be in instrument units also.
>
> A simulation for 10 mg/ml of a 5 kDa molecule at 60K (assuming vbar = .73
> and rho = 1) should not reach 250 fringes at the cell base. For such a
> simulation I get about 150 fringes at the base, and ~30 fringes at a
> location ~60% of the way down the cell, which should indeed be close to
> the loading concentration of 33 fringes. If you are getting 250 fringes
> at the cell base I suspect you did not change the mass from the default
> 68 kDa.
>
> Also, regarding Jo Butler's comment, while he is certainly right that the
> refractive increment is also wavelength dependent, I beg to differ with
> his statement that the wavelength does not directly influence the fringes
> per mg/ml conversion factor.
>
> 'Hope this helps,
>
> John Philo
>
> -----Original Message-----
> From: rasmb-admin at server1.bbri.org
> [mailto:rasmb-admin at server1.bbri.org]On Behalf Of Kristian Schilling
> Sent: Tuesday, October 23, 2001 9:41 AM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] RE: still Beckman software
>
>
> The older archived RASMB emails can be found at:
>
> http://rasmb-email.bbri.org/rasmb_archives
>
> and current archives at
>
> http://rasmb-email.bbri.org/pipermail/rasmb/
>
> -------------------------------------------------------------------------
> --- ------
>
> John,
>
> have you actually seen this formula work quantitatively? In the sense,
> perhaps, that the fringe number you get in the simulation actually matches
> fringes measured for that material.
>
> The factor of 3.3 you give would fit for 1,2 cm pieces, 675 nm light and
> dn/dc = 0.190 ml/g, it would be in mg/ml. If I do a simulation with
> meniscus depletion, say a 5000 Da species @ 60k, I get about 250 fringes
> at the cell bottom for 10 mg/ml. There is no Ja here, so it is fairly
> easy to calculate an initial loading concentration, which would be about
> 150 fringes. Such a sedimentation boundary would mean 44 mg/ml under the
> given conditions. And not 10.
>
> Either I am doing something terribly wrong or there is some additional
> factor in there.
>
> Kristian (not yet happy)
>
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P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296



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