[RASMB] Band Sedimentation

[Jacob Lebowitz]

David,

A recent short description of band centrifugation was published by us in 
the Biochemical Society Transactions (1998) 26, 745-749.  I assume that you 
have a band centerpiece with a channel to the sector.  Assemble the bottom 
window and add the band centerpiece. Then fill the well with 20 ul of 
protein solution. A concentration of 1 mg/ml for detection at 280nm should 
work well. You can decrease the concentration if you want to use 230nm. 
After filling the well complete the assembly of the cell as for a boundary 
run. Possible problems that you may have encountered are as follows: 1. If 
you over fill the sector with buffer solution you can trap the protein 
solution in the well.  Hence, I use 350 ul of buffer solution which allows 
layering of the well solution on top of the sector solution; 2. Yes, it is 
critical to have a sufficient density difference between the well solution 
and the column solution. A diffusion density gradient needs to be 
established that prevents convection.  If the protein can be in 0.1X PBS 
then a 1X PBS solution will generate a stabilizing gradient (see Fig.1 of 
the above paper).  While the latter works we often can not dilute the 
protein so we use 50% D2O:H2O PBS for the sector solution. This works well 
for almost all cases. The density of this solution is 1.0527 which is 
generally much greater than most common buffer solutions. However, if you 
have a protein in high salt it could be possible that you will have 
insufficient density gradient and convection will occur and protein will 
transfer through the cell to the bottom, i.e. no band will form. You should 
be safe using  50% D2O:H2O PBS which also has a relative viscosity of 
1.1295. You can increment in the contributions of your buffer in order to 
correct to s20,w. For data analysis you can use Peter Schuck's Sedfit which 
can fit band data. For a single component good fits require fairly clean 
material. If other components are in the band that are not separated you 
can use the Peter's c(s) vs. s analysis of the data. Alternatively, there 
is a simpler program that Peter developed called Sedband which we can send 
you if you want it. It works like Sedfit. However, only the weight average 
s will be obtained. Let me know if new band experiments are successful. We 
need to get all the above into the literature for investigators but other 
pressing projects keep delaying writing a band methodology paper.

Jack Lebowitz

At 01:38 PM 2/26/02 +0000, you wrote:
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>Hello all
>
>Does anyone have a protocol for using a band forming centrepiece?
>
>I seem to remember that the buffer solution you are forming a band in
>needs to be denser than the solution you wish to analyse, however trial
>runs have been, to say the least, unconvincing.
>
>Thanks in advance
>
>Dr. David J. Scott
>Department of Biochemistry
>School of Medical Sciences
>University of Bristol
>Bristol BS8 1TD
>United Kingdom
>Phone: +44 (0)117 954 6873
>Fax:   +44 (0)117 928 8274
>Email: david.scott at bristol.ac.uk
>Web:   http://www.bch.bris.ac.uk/staff/halford/djswebpage.htm
>
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Jacob Lebowitz, PhD
Molecular Interactions Resource
Division of Bioengineering and Physical Science, ORS
National Institutes of Health
Mail: Bldg. 13 Rm. 3N17
Office Bldg. 13 Rm. 3E49
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1955
Fax: (301) 480-1242
email: lebowitz at helix.nih.gov