[RASMB] higher Mw limit of analytical ultracentrifugation

[Jacob Lebowitz]

Joris,

Another possibility for detecting the binding of the 30 kDa protein to the 
large multimer-protein is to monitor the decrease in absorbance of the 
small protein caused by the binding to the large protein using 
sedimentation velocity. The larger protein would sediment rapidly away from 
the 30 kDa protein and binding would decrease  the absorbance from the 
initial loading value of the 30 kDa protein. I remember a Biochemistry 
paper by Rezvin (I am almost sure, perhaps someone else can help)
  in the late 1980s that measured the association constant of core RNA 
polymerase to very large DNA using this approach.  By titrating in 
different amounts of the smaller component and measuring the amount bound 
by the decrease in absorbance and hence the concentration of free protein 
ligand Rezvin could obtain Scatchard plots.

Jack Lebowitz




At 04:57 PM 6/30/2004 +0200, Joris Beld wrote:
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>Dear all,
>
>One of my colleagues asked me to measure a rather heavy multimer-protein
>complex and I was wondering what the higher molecular weight limit of an
>XL-A analytical ultracentrifuge is. I found in some literature that 1.0 MDa
>is considered the maximum. My particular protein-multimer has a Mw around
>1.3 MDa and forms a complex with a protein of 30 kDa. Can one see the
>equilibrium (or just association) of the small protein with the big protein
>multimer? Can one distinguish the small protein and the multimer? If someone
>has some information on this, I would appreciate any help since I'm pretty
>new at analytical ultracentrifugation.
>
>Thanks in advance.
>
>Kind regards,
>
>Joris Beld
>
>(btw, thank you all for the comments on the torque wrench spare part;
>unluckily I already ordered one from Beckman (400$); but next time we'll try
>to get the mechanical workshop to make one from a saw blade)
>
>--
>Joris Beld
>Laboratory of Organic Chemistry
>ETH-Hönggerberg
>HCI-F322
>CH-8093 Zürich
>Switzerland
>+ 41 1 6322972
>beld at org.chem.ethz.ch
>http://www.protein.ethz.ch
>--
>
>
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