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<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff>David,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff>Regarding filling the cell all the way up to the top, it isn't
just a number of data points issue, it's primarily a separation issue. The
longer the solution column, the more physical separation of different sized
aggregates you will achieve in your samples. This is because separation is
proportional to time while diffusional spread is proportional to the square root
of time - longer solution columns facilitate increased total separation.
Having that extra separation improves the ability to resolve aggregates much
more than the simple increase in number of data points. This is
particularly important if you are trying to separate an irreversible dimer
aggregate from the monomer.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT color=#0000ff>Also,
I really don't see any reason to worry about non-linearity in pipetting for
filling the cell. We simply dial our p200 to about 230 uL and add this
volume twice, and this gets us all the way up to the top of the solution
column. Who cares if 228 uL are delivered instead of 230 uL; we only
care about consistent loading and getting as much in the solution column as
possible. Of course, accurate pipetting is paramount for dilutions, but
that's not what we are considering here... </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT color=#0000ff>Also,
if you run into leaking issues during loading - due to capillary action drawing
the liquid out of the cells - in my experience this arises primarily from
wetting the inside of the tiny filling holes from multiple insertions of the
pipette tip. To avoid this, my standard loading procedure is to insert a
free gel loading tip into the filling hole and leave it there - this keeps the
filling hole dry. I then put another gel loading tip onto my p200 and add
the solution by inserting the tip attached to the p200 into the free
tip I already placed into the filling hole. Gentle pressure makes a
nice seal that allows you to pipette the contents of the top tip through
the bottom tip into the interior of the cell. To avoid trying to
pipette against a vacuum, I lift the bottom tip up a bit with one
hand, then pipette with the other. In this way you can load into the
bottom tip as many times as you want and not wet the inside of the filling
hole. The other important issue is to load slowly to try to avoid the
sides of the solution "crawling up the windows" as the sample is loaded due to
capillary action. This procedure is especially useful for loading solutions that
contain high concentrations of detergents.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff>Regards,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff>Karl</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=261330115-21102015><FONT
color=#0000ff></FONT></SPAN> </DIV><BR>
<DIV lang=en-us class=OutlookMessageHeader dir=ltr align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> RASMB
[mailto:rasmb-bounces@list.rasmb.org] <B>On Behalf Of </B>David
Hayes<BR><B>Sent:</B> Tuesday, October 20, 2015 6:03 PM<BR><B>To:</B>
RASMB<BR><B>Subject:</B> Re: [RASMB] Strange AUC noise<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV
style="FONT-SIZE: 16px; FONT-FAMILY: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, sans-serif; COLOR: #000; BACKGROUND-COLOR: #fff">
<DIV id=yui_3_16_0_1_1445386549717_7766><SPAN
id=yui_3_16_0_1_1445386549717_7799>Hi Tom and all,</SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_7985><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_8161 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>The practice in our lab is to use Spin50
cells always for absorbance scanning since even at best it seems that Spin60
cells always have a higher RMSD for absorbance scanning (maybe a bit less signal
to noise due to being narrower, or because of the timing issues, don't know,
just observed). Since we had been averaging about 600 samples a year, we
have other practices not found in the best academic labs. I instruct my
analysts to load 400 microLiters on the sample side and 405 - 410 on the
reference side. We always use gel loading pipettes for loading to avoid
scratching the walls with needles. We use Beckman supplied thin tubing or
pipette tips to remove the sample rather than blunt needles, also to avoid wear
and tear on the centerpieces. Given that some samples are precious and we
are always given only enough for one filling, and some samples are
irreplaceable, trying to fill up closer to the top risks over filling spills
both by getting the bubble too close to the top and by dipping the pipette tip
in three times since most of our pipettes get a bit non-linear over 200 micro
liters. And since the main data we want is percent aggregate, and we want
to avoid as much as possible any possible artifacts, we use absorbance data only
and are very conservative in choosing the area to fit fit. Empirically I
am of the opinion that there are plenty of data points, and another mm of data
is not going to be very important to the SEDFIT final answer. Jack has
mentioned to me before, and stated in this email chain, that his practice is to
insist on analysts optimizing the column height in the cells to near the max
possible, which makes sense theoretically, but practically I don't think the
extra data points change the final answers enough to justify the extra stress
and time taken by the analysts to try to achieve that optimization. I get
much more bang for the nagging by telling them to be more careful washing their
cells: I have observed noticeable differences in final answers between
dirty and clean cells when doing replicates, and also when the lamp is cleaned,
I sometimes resolve peaks better in certain samples.<BR></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_7800><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_7844 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>At first the Beckman repair person did not
see anything wrong with the slit assembly, but later after I wrote the email to
RASMB the instrument failed a radial calibration and he did replace the slit
assembly. Subsequently, the instrument is now running OK
again.</SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_7903 dir=ltr><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_8323 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>However, the last time we saw noise like
this, replacing the slit assembly helped for only a few weeks and then the noise
came back, so I am not completely convinced there is nothing else going on
(The last time we saw this kind of noise, quite a few parts were changed for
reasons that are a bit complex and can't be put in the email). The noise
is never seen in the scan of a blank hole without a cell, so maybe it is both
the slit assembly and something with the timing and cell light beam
geometry. We also had a slit assembly become unglued in our oldest
centrifuge and give the "jumping pattern" noise John Philo described (as well as
finding the bottom of the cell to be outside the normal 7.15 to 7.2 cm range)
but none of our older machines have made this exact strange
pattern.</SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_8943 dir=ltr><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_8942 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>Thanks for the discussion. If there are
any new developments, I will update the conversation. For now, changing
the slit assembly has fixed the problem and prevents further
investigation: an unexpected good news, since last time changing out the
assembly provided only limited help.</SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_9040 dir=ltr><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_9166 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>Kind Regards,</SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_9165 dir=ltr><BR><SPAN
id=yui_3_16_0_1_1445386549717_7799></SPAN></DIV>
<DIV id=yui_3_16_0_1_1445386549717_9145 dir=ltr><SPAN
id=yui_3_16_0_1_1445386549717_7799>David</SPAN></DIV><BR>
<DIV id=yui_3_16_0_1_1445386549717_7769
style="FONT-SIZE: 16px; FONT-FAMILY: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, sans-serif">
<DIV id=yui_3_16_0_1_1445386549717_7768
style="FONT-SIZE: 16px; FONT-FAMILY: HelveticaNeue, Helvetica Neue, Helvetica, Arial, Lucida Grande, sans-serif">
<DIV id=yui_3_16_0_1_1445386549717_7767 dir=ltr>
<HR id=yui_3_16_0_1_1445386549717_7771 SIZE=1>
<FONT id=yui_3_16_0_1_1445386549717_7770 size=2 face=Arial><B><SPAN
style="FONT-WEIGHT: bold">From:</SPAN></B> "Laue, Thomas"
<Tom.Laue@unh.edu><BR><B><SPAN style="FONT-WEIGHT: bold">To:</SPAN></B>
"jphilo@mailway.com" <jphilo@mailway.com>; 'David Hayes'
<drdavidbhayes@yahoo.com>; 'RASMB' <rasmb@rasmb.org> <BR><B><SPAN
style="FONT-WEIGHT: bold">Sent:</SPAN></B> Tuesday, October 20, 2015 8:54
AM<BR><B><SPAN style="FONT-WEIGHT: bold">Subject:</SPAN></B> RE: [RASMB] Strange
AUC noise<BR></FONT></DIV>
<DIV class=y_msg_container><BR>
<DIV id=yiv7929042017>
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<DIV class=yiv7929042017MsoNormal><SPAN style="FONT-SIZE: 11pt">Hi-</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal><SPAN style="FONT-SIZE: 11pt">Were these
Spin60 or Spin50 cells? If they were Spin60, it might be that the lamp timing on
the ‘noisy’ machine is causing the light pulse to partially hit the centerpiece
wall. This is a machine-to-machine problem, consistent with what you are seeing.
However, the telltale for the timing problem is increased noise in the scans,
often at the top of the cell, not a wave. Consequently, I vote for the
slit assembly lifting (John’s suggestion).</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal><SPAN style="FONT-SIZE: 11pt">Tom</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal><SPAN
style="FONT-SIZE: 11pt"></SPAN> </DIV>
<DIV class=yiv7929042017MsoNormal><SPAN
style="FONT-SIZE: 11pt"></SPAN> </DIV>
<DIV class=qtdSeparateBR><BR><BR></DIV>
<DIV id=yiv7929042017yqt00390 class=yiv7929042017yqt7125105686>
<DIV>
<DIV
style="BORDER-TOP: #b5c4df 1pt solid; BORDER-RIGHT: medium none; BORDER-BOTTOM: medium none; PADDING-BOTTOM: 0in; PADDING-TOP: 3pt; PADDING-LEFT: 0in; BORDER-LEFT: medium none; PADDING-RIGHT: 0in">
<DIV class=yiv7929042017MsoNormal><B><SPAN
style="FONT-SIZE: 10pt">From:</SPAN></B><SPAN style="FONT-SIZE: 10pt"> RASMB
[mailto:rasmb-bounces@list.rasmb.org] <B>On Behalf Of </B>John Philo<BR
clear=none><B>Sent:</B> Monday, October 19, 2015 5:00 PM<BR
clear=none><B>To:</B> 'David Hayes'; 'RASMB'<BR clear=none><B>Subject:</B> Re:
[RASMB] Strange AUC noise</SPAN></DIV></DIV></DIV>
<DIV class=yiv7929042017MsoNormal> </DIV>
<DIV class=yiv7929042017MsoNormal style="MARGIN-BOTTOM: 6pt"><SPAN
style="FONT-SIZE: 10pt">David, I will speculate that you have a problem with the
slit assembly. You might see something like that if the foil on the bottom side
has come partially loose. Or perhaps there is some dirt or a burr causing the
whole assembly to lift up at certain radial positions.</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal style="MARGIN-BOTTOM: 6pt"><SPAN
style="FONT-SIZE: 10pt">I once had the foil come loose in a way that produced
wild outlier scans, but only every once in while (a few per run). Intermittently
the foil would actually catch on something (probably the ring on top of the PMT)
and fold completely back on itself.</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal style="MARGIN-BOTTOM: 6pt"><SPAN
style="FONT-SIZE: 10pt">And by the way, you should remind your people to fill
the cells <U>much</U> more. You want the meniscus to be up near 5.9 cm, not at
~6.25 cm.</SPAN></DIV>
<DIV class=yiv7929042017MsoNormal style="MARGIN-BOTTOM: 6pt"><SPAN
style="FONT-SIZE: 10pt">John</SPAN></DIV>
<DIV>
<DIV
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<DIV class=yiv7929042017MsoNormal><B><SPAN
style="FONT-SIZE: 11pt">From:</SPAN></B><SPAN style="FONT-SIZE: 11pt"> RASMB [<A
href="mailto:rasmb-bounces@list.rasmb.org" shape=rect rel=nofollow target=_blank
ymailto="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</A>]
<B>On Behalf Of </B>David Hayes<BR clear=none><B>Sent:</B> Monday, October 19,
2015 11:14 AM<BR clear=none><B>To:</B> RASMB <<A
href="mailto:rasmb@rasmb.org" shape=rect rel=nofollow target=_blank
ymailto="mailto:rasmb@rasmb.org">rasmb@rasmb.org</A>><BR
clear=none><B>Subject:</B> [RASMB] Strange AUC noise</SPAN></DIV></DIV></DIV>
<DIV class=yiv7929042017MsoNormal> </DIV></DIV>
<DIV>
<DIV id=yiv7929042017yqt11234 class=yiv7929042017yqt7125105686>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2742>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>Hi
all,</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2742>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>We had some
strange AUC noise in a recent experiment. In the pictures below from
SEDFIT, the first two runs produced data as in the picture NNBekman1 (the IT
guys name our instruments and they pruposely mispelled Beckman as Bekman years
ago). A strange noise pattern showed up in the scans that were produced on day
one. Then the sample was loaded into a new cell and run again on the same
centrifuge producing the strange noise pattern in the picture called NNBekman1.
the filled cell was taken and gently mixed and then run on another
centrifuge Bekman17 and made a perfectly fine patter shown in picture
Beckman17.</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>We have seen
the strange noise before on our NNBekman1 centrifuge, but this is the first time
on NNBekman2.</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>Has anyone
seen noise like this? </SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>Any theories
on the cause of the noise?</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>It certainly
looks like an optical artifact of some kind, but the time dependence is hard to
explain.</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>Kind
Regards,</SPAN></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal style="BACKGROUND: white"><SPAN>David
Hayes</SPAN></DIV></DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2751>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2742>
<DIV class=yiv7929042017MsoNormal
style="BACKGROUND: white"><SPAN></SPAN> </DIV></DIV>
<DIV id=yiv7929042017yui_3_16_0_1_1445277124881_2742>
<DIV class=yiv7929042017MsoNormal
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