[RASMB] Problems in analysis of proteins using analytical ultracentrifugation

[Laue, Thomas]

Hi Prasad-
To a reasonable first approximation, vbar is the inverse of the density of protein (~1.4 g/ml), and does not change with size. If you sediment at very low speeds, you may be able to see the boundary going down the cell in buffer. You also may wish to increase the solvent viscosity using, for example, gycerol to slow down the sedimentation. The sedimentation coefficient will decrease as 1/viscosity, which will allow you to get more scans before the protein aggregates (micelles, perhaps?) pellet. If there is concern that the glycerol is affecting the aggregation, you can repeat the experiment at a few glycerol concentration, correct the data for the viscosity/density of the solvent back to s20,w and see if the g(s) distributions change appreciably.
What problems are you having with the lipoprotein? If you are seeing aggregation, you may need to add a detergent to 'cover' the lipid tails. Which detergent to use becomes trial and error black magic... unless someone has experience with your protein.
Best wishes,
Tom
________________________________
From: RASMB [rasmb-bounces at list.rasmb.org] on behalf of Prasad Satyamurthy [psatyamurthy at wockhardt.com]
Sent: Friday, December 05, 2014 1:49 AM
To: rasmb at rasmb.org
Subject: [RASMB] Problems in analysis of proteins using analytical ultracentrifugation

Dear All,
             I have couple of problems in the analysis my proteins using analytical ultracentrifugation.
First one is that I am trying to analyse the association state of my protein complex that is formed in between two proteins that associate to form crystals in buffer solutions. These crystals are about a few microns in size. So it is possible to study the association state of such big size particles using analytical ultracentrifugation. Also I do not know how to calculate the v bar of bigger size particles of about 500 nm is it independent of size?

Second problem that I was having was when I was analysing a lipoprotein (protein having a hydrophobic lipid tail attached to it) does any one have any idea how to analyse such samples ?

Any opinion or suggestion regarding the above problems that I am facing would be of great help to me.


Thanks



Prasad Satyamurthy

Genomics and Biotechnology

D4 Chikalthana Aurangabad

Email: psatyamurthy at wockhardt.com


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