[RASMB] Centerpiece question

Fri Dec 19 06:24:40 PST 2014

Hi John and Walter,
I think that Walter has misnamed the cells.  The SedVel50 use the standard width middle rib and have a total of 500 uL space in each sector which uses the 440 and 420 uL to leave a reasonable air space.  The SedVel60 cells have a wider middle rib and have smaller total volumes, and I think that it was around 330 and 310 uL for meniscus matching.  While I was at Walter's lab we had a few SedVel50's and one SedVel60 for testing, but we still used mostly Walter's homemade meniscus matching cells.
David
      From: Walter Stafford <wstafford3 at walterstafford.com>
 To: John Sumida <jpsumida at U.WASHINGTON.EDU> 
Cc: rasmb at rasmb.org 
 Sent: Friday, December 19, 2014 2:46 AM
 Subject: Re: [RASMB] Centerpiece question

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:

#yiv1694068238 #yiv1694068238 -- _filtered #yiv1694068238 {font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;} _filtered #yiv1694068238 {font-family:Tahoma;panose-1:2 11 6 4 3 5 4 4 2 4;}#yiv1694068238 #yiv1694068238 p.yiv1694068238MsoNormal, #yiv1694068238 li.yiv1694068238MsoNormal, #yiv1694068238 div.yiv1694068238MsoNormal {margin:0in;margin-bottom:.0001pt;font-size:11.0pt;}#yiv1694068238 a:link, #yiv1694068238 span.yiv1694068238MsoHyperlink {color:blue;text-decoration:underline;}#yiv1694068238 a:visited, #yiv1694068238 span.yiv1694068238MsoHyperlinkFollowed {color:purple;text-decoration:underline;}#yiv1694068238 p.yiv1694068238MsoAcetate, #yiv1694068238 li.yiv1694068238MsoAcetate, #yiv1694068238 div.yiv1694068238MsoAcetate {margin:0in;margin-bottom:.0001pt;font-size:8.0pt;}#yiv1694068238 span.yiv1694068238BalloonTextChar {}#yiv1694068238 span.yiv1694068238EmailStyle19 {color:windowtext;}#yiv1694068238 span.yiv1694068238EmailStyle20 {color:blue;font-weight:normal;font-style:normal;text-decoration:none none;}#yiv1694068238 span.yiv1694068238EmailStyle21 {color:blue;}#yiv1694068238 .yiv1694068238MsoChpDefault {font-size:10.0pt;} _filtered #yiv1694068238 {margin:1.0in 1.0in 1.0in 1.0in;}#yiv1694068238 div.yiv1694068238WordSection1 {}#yiv1694068238 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 SumidaUniversity 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 questionJohn, 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 FacilityUniversity of WashingtonMolecular Engineering & Sciences Institute, G223946 West Stevens Way NESeattle WA 98195-1653 _______________________________________________
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