[RASMB] higher Mw limit of analytical ultracentrifugation

[Peter Schuck]

Hi Joris,
we've had a similar problem recently, binding of a 24 kDa protein to a ~ 
MDa complex (heterogeneous mixture of big stuff, actually).  Our question 
was the stoichiometry of binding.  We did a sedimentation velocity run and 
looked at the changes of the s-value of the big complex.  That worked very 
well, because there were many sites and we saw a significant shift.  If it 
would have been a 1:1 binding between the small protein and the big 
complex, the sedimentation velocity probably would not have worked unless 
you could specifically detect the small protein.

However, there are other approaches that might work well.  I haven't done 
that, but would expect it should work well:  In principle if you use a 
rather low rotor speed, you have a problem similar to that of small 
molecule (few hundred Dalton) to ordinary sized proteins.   (Probably one 
should use not too long solution columns and give it a few days to reach 
equilibrium. )  I would try sedimentation equilibrium with mass 
conservation constraints, or multi-wavelength detection if you can put a 
chromophoric label on the smaller protein.  Basically, what one would 
detect is not the change in mass of the complex (that's unlikely to be 
possible), but the loss of free concentration of the small protein.

Michelle Arkin and Jim Lear have described some techniques for that recently
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11726190
and, some equilibrium analysis models with mass conservation are 
implemented in SEDPHAT.

Peter


At 04:57 PM 6/30/2004 +0200, you 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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