[RASMB] RASMB Digest, Vol 34, Issue 4

Wed Jul 11 00:48:09 PDT 2012

Dear All,
             I am a PhD student at Paul Scherrer Institute , Switzerland.  I have a problem with a protein which I am trying to analyze using Sedimentation Velocity ( absorbtion detection system ) for determining the monomer and dimer fractions and thus the Kd. 

             I am doing the run at 37°C at 40000rpm using the Beckmann centerpieces. But I am having problem with aggregration and thereby the initial scans clearly show something very big sedimenting out. However I have measured the spectra and folding / refolding curve of the protein in CD and also looked for the scattering signal change at 600nm over 20hrs. The protein folds back completely and has a high alpha helical content. The scattering signal changewould be about 12% of the total signal after 20hrs.
I have no clue what is going on. The same protein with a GFP tag behaves fine at 22°C. So I am wondering what is reall ygoing on.
Please could someone help me out. I could provide additional information if needed. It would be really great if somebody has some suggestions.

Thank you ,
Regards
Indrani Sen

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Sent: 07 July 2012 18:00
To: rasmb at rasmb.bbri.org
Subject: RASMB Digest, Vol 34, Issue 4

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Today's Topics:

   1. Density increment implications? (John Sumida)
   2. Announcement: SEDFIT/SEDPHAT workshop in Japan,	September
      2012 (Susumu UCHIYAMA)

----------------------------------------------------------------------

Message: 1
Date: Fri, 6 Jul 2012 23:44:13 -0700
From: "John Sumida" <jpsumida at gmail.com>
To: <rasmb at rasmb.bbri.org>
Subject: [RASMB] Density increment implications?
Message-ID: <001401cd5c0b$ecaf75d0$c60e6170$@gmail.com>
Content-Type: text/plain; charset="us-ascii"

Dear RASMB,

Thanks to everyone who responded to my previous question regarding the linearity of density and concentration.  Although I am not completely resolved in this yet, for the time being I've decided to work in a range where our measurements appear to be linear and thus reasonable and understandable.  All the comments received were, as always highly informative and very helpful.  

I have another question along the same lines.  According to a lot of previous work it does seem that it is accepted given that  v-bar is, for the most part, a constant for most proteins, and that the density is likewise a constant at a given solution composition and temperature, the density increment, (1-vp), is therefore also accepted to be constant.  

Two questions:

1.       Am I correct in thinking this? 

2.       Can the density increment of a solution be used to convert the
density to concentration as it is stated in "Adair et. al. Density increments of proteins. Proc. Royal Soc. Lond, A Math Phys Sci 1947 pp.190_34"?

Thank you again for all the helpful pointers.

John Sumida, Ph.D.

Analytical Biopharmacy Core

University of Washington

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Message: 2
Date: Sat, 07 Jul 2012 17:37:07 +0900
From: Susumu UCHIYAMA <suchi at bio.eng.osaka-u.ac.jp>
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Announcement: SEDFIT/SEDPHAT workshop in Japan,
	September 2012
Message-ID: <4FF7F533.1000001 at bio.eng.osaka-u.ac.jp>
Content-Type: text/plain; charset="iso-2022-jp"

Dear Colleagues,

This is the second announcement for SEDFIT/SEDPHAT workshop in Japan, September 2012.
We will have two workshops in Tokyo Institute of Technology, Japan.
The workshop is held in English and includes training session by instructors.  

1. September 8-12, 2012
Asian Workshop on Hydrodynamic and Thermodynamic Analysis of Biological Macromolecules with SEDFIT and SEDPHAT 2. September 13, 2012 Asian SEDPHAT Workshop: A workshop focused on ITC analysis

Please refer detailed information at
http://www.biomoleculesinteractions.com/sub1.html
http://www.biomoleculesinteractions.com/sub4.html

Hope to see many of you there,
Susumu

Susumu Uchiyama, PhD
Associate Professor
Department of Biotechnology
Graduate School of Engineering
Osaka University
2-1 Yamadaoka, Suita,
Osaka, 5650871, Japan

phone: 81-6-6879-4216
email: suchi at bio.eng.osaka-u.ac.jp

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