[RASMB] Centerpiece question

Walter Stafford wstafford3 at walterstafford.com
Thu Dec 18 23:46:15 PST 2014


Hi John,
	The maximum volume depends on whether you are using the SedVel50 or SedVel60 version.You have to fill the sides so that the matched menisci (i.e. the sample and the reference menisci) are both above the capillary (the one that starts on the reference side, runs down the middle of the center rib and into the bottom of the sample side).
Otherwise, the reference buffer will stop flowing to the sample side when the meniscus gets to the capillary and too little buffer will have been transferred. And the menisci will not be matched.

You will have to experiment a little to find the sweet spot. I was using the SedVel60 version and - I may not be remembering correctly but I think I used 440 uL on the reference side and 420 uL on the sample side. So that when matched there was 430 uL on each side and my menisci were at 5.90 cm. This gives the longest sedimentation path, if you will. It's wasteful to use a smaller volume: the longer sedimentation path gives you more resolution. Each run is expensive, so you might as well get the most out of it.

Walter
On Dec 18, 2014, at 14:23, John Sumida <jpsumida at U.WASHINGTON.EDU> wrote:

> Dear RASMB
>  
> Thank you for your responses to my question.  For the Stafford meniscus matching centerpieces what is the max volume that you can fill these centerpieces to?
>  
> Best regards,
> John Sumida
> University of Washington
>  
> From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Karl Maluf
> Sent: Thursday, December 18, 2014 11:03 AM
> To: rasmb at rasmb.org
> Subject: Re: [RASMB] Centerpiece question
>  
> The other issue with the meniscus matching centerpeices is to be careful to not overfill the buffer channel higher than the top capillary.  This prevents equalization of the pressures between the two channels, and can inhibit the desired meniscus matching.
>  
> Karl
>  
> From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Philo
> Sent: Thursday, December 18, 2014 10:49 AM
> To: 'John Sumida'; rasmb at rasmb.org
> Subject: Re: [RASMB] Centerpiece question
> 
> John, in my view the advantages of the Stafford meniscus-matching design (available from Spin Analytical) over the synthetic boundary centerpieces are that the long capillary design inhibits diffusion between the two channels and any inadvertent fluid transfer during loading and handling. The drawback of that long channel though is that it is harder to get the transfer needed to match the heights (it takes more force)---I find I may need to go to 10-12 K rpm. For some of the virus samples we run that is >= to the run speed, so you have to do it very quickly to avoid significant sedimentation during the meniscus matching spin.
>  
> John
>  
> From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
> Sent: Thursday, December 18, 2014 9:45 AM
> To: rasmb at rasmb.org
> Subject: [RASMB] Centerpiece question
>  
> Dear RASMB,
>  
> For interference measurements where matching the sample and reference channel menisci is essential, is there a preference for either the meniscus matching centerpiece versus synthetic boundary centerpiece.  We would be interested to hear about pros and cons.
>  
> Thank you and happy holidays.
>  
> Best regards,
>  
> John Sumida, Ph.D.
> Analytical Biopharmacy Core Facility
> University of Washington
> Molecular Engineering & Sciences Institute, G22
> 3946 West Stevens Way NE
> Seattle WA 98195-1653
>  
> _______________________________________________
> RASMB mailing list
> RASMB at list.rasmb.org
> http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org



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