[RASMB] Protocol for Gravitational Sweep Sedimentation Velocity Study

[John Correia]

James
 
I have recently run gold and find it to be very big with a broad distribution from 100 s to 300 s.  It is very condition dependent as well, especially the presence of divalent cations.  Find a speed where you see sedimentation and analyze those data 1st before you try to design a sweep protocol.  Sweep is designed for distributions that vary by multiple log units.  DCDT works fine for analysis of this kind of broad data.  However, if you find its still too big switch to a narrower radial scan range that collects data just in the center of the cell, eg 6.45 to 7.05 cm.  Sedanal has a wide distribution analysis program embedded in the DCDT analysis section for this kind of data.  Note for really big stuff you can only run one sample as a time with the XLA, allowing you to collect as much data as possible during the rapid sedimentation.  And you will need to do a scan later to determine the meniscus positions at each speed.  The procedure is described in Biophys Chem 108, 273, 2004.  
 


>>> On 5/5/2009 at 6:20 PM, in message <7E80450DE7474E4881E405C742AC0DEA0A6C2D0449 at MBCLUSTER.xchange.nist.gov>, "Falabella, James" <james.falabella at nist.gov> wrote:

Dear RASMB Members,
 
I am performing sedimentation velocity to examine the particle size distribution of 10 nm diameter gold particles that I have intentionally induced into stable aggregates. I suspect that the aggregates population ranges from dimers to tetramers. At first, I tried to sediment the aggregated particles at 7000 RPM in an XL-A outfitted with: 4-hole ANTI60 rotor, 12 mm charcoal-Epon cells, quartz windows and the absorbance scans at 520 nm (with the 400 nm filter in the light path) appeared mal-formed while the scans of another cell with the non aggregated 10 nm diameter gold particles are well formed sigmoids and easily fit by Sedfit. I looked up some references from the literature on the analysis of wide distribution samples with an analytical ultracentrifuge that were very good background, however, they did not have a protocol that spelled out what buttons to press to get the XL-A to collect the data at different angular velocities. I went ahead with my centrifugation study by performing several back to back runs where I simply started run after run using the Beckman software to increase the rotor speed from 3000 to 5000 to 8000 RPM, however, the 5000 and 8000 RPM runs could not be fit by Sedfit due to the fact that there was not a long enough acceleration phase from one speed to the next.  I looked through Sedfit and could not find a way to get around this error and it is probably not a good idea to try my own fix before asking around for any tips on how to address the proper operation of the XL-A for a gravitational sweep and data analysis.
 
Thank you in advance for your advice,
 
James Falabella 


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