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Hi fellow RASMB'ers<br><br>
I have a set of sedimentation velocity experiments in which I have become
suspicious of non-ideality and convection, or both.<br><br>
I am trying to collect information on convection and also want to put out
some theoretical ideas for your consideration or even your amusement,
since convection is so annoying that I am willing to consider and will
present any crazy idea if it might possibly help.<br><br>
First, I have seen some data sets where the boundary shape (especially
seen by dcdt methods or ls-g*(s)) is obviously asymmetric and just looks
odd. This is usually attributed to convection and sometimes seems
correlated to a cell with a bent centerpiece or to improper cell
alignment in the rotor. The usual procedure here is to just repeat
the experiment with new cells or better alignment and throw out the bad
data. In this case, convection is just an annoying problem that
makes more work.<br><br>
However, in the case of this data, the peaks do not look obviously
asymmetric, they fit to a single main peak most of the time (some double
peaks in c(s) occurred) and repeated experiments in different cells just
keep varying. I have fit the data with Sedanal and with Sedfit and
am convinced that the data sets from the repeated experiments are
actually different and that the variability is not an artifact of the
fitting process and the random noise on the data. <br><br>
So the conjecture I am considering right now is this: Since c(s) in
Sedfit models all aspects of the data as either one of the noises
(baseline, TI, RI) or as an amplitude of a single ideal species among
other non-interacting species, wouldn't c(s) report small amounts of
convection as a species in the cell since convection is not a baseline,
is not time invariant, and is not radially invariant? This may
explain the variability in the repeated experiments I am looking
at. For whatever reason, these samples are prone to small amounts
of convection and that is why they do not reproduce as well as they
should.<br><br>
How is convection identified?<br>
Peter Schuck implied that when the initial scans do not fit well, you may
suspect convection.<br>
Excess broadening of the boundary at the meniscus could be due to
convection. With curve fitting, this may result in the meniscus
fitting to location closer to the center of the rotor than it obviously
is by optical inspection. <br>
Tom Laue has said that convection "follows the cell". If
the is cell is refit into another whole and the non-reproducible result
follows the cell, suspect convection.<br>
Looking at data with Walter Stafford using g(s), he also pointed out that
when there is a bumpy leading edge that does not seem to move as a well
fit boundary (different sets of scans give different S values) you can
suspect the bumps are not real boundaries but convection.<br>
Obviously asymmetric boundaries that have bumps or peaks in g(s) that are
too steep or that change shape drastically (diffusion makes Gaussian
curves or the sum of Gaussian curves that are pretty smooth) suspect
convection.<br>
Are there any other tell-tale signs?<br><br>
What causes convection.<br>
Tom Laue wrote a bit on this before in the Sedfit mailing list: I
will past that here so he won't have to re-write it for RASMB.<br>
1- Probably the most common cause of convection is damage (usually a
scratch from a needle tip) to the channel walls. Inspect the walls
carefully. If there are any scratches, the centerpiece should be
replaced.<br>
2- The second most common problem is a warped centerpiece septum. One way
to detect this is to place the suspect centerpiece over a
"good" centerpiece and look to see if there is any place where
the two septums do not line up. If you have a tool makers microscope, you
can line up the centerpiece septum and look to see if the wall can be
aligned with the scribe lines on the microscope viewer. The warping
usually occurs if the cell has leaked on one side but not the other.<br>
3- The third most common problem is the build up of junk on the walls.
You will see this when you inspect the centerpiece- the walls will look
hazy or have a spot or spots on them. If you have been working with
proteins, sometimes they will dry onto the wall and be very difficult to
remove. Your best bet in this case is to soak the centerpiece overnight
in a solution of pepsin (~1 mg/ml) in 1 mM HCl. Then wash the cell
thoroughly with soap and water, then water then a drying agent (dry N2-
my choice, EtOH or acetone). This problem usually crops up if a
centerpiece is very old or if it has held a high concentration protein
solution.<br>
4- The fourth most common reason is temperature instability at the start
of a run. Be sure the temperature is stable before starting a velocity
run.<br>
Peter Shuck mentioned that for him the temperature instability was
critical.<br><br>
How to prevent convection.<br>
1. Throw away damaged cells and treat the new ones gently but
cleanly.<br>
2. Make sure you have temperature equilibrium.<br>
3. Walter Stafford mentioned that ramping the rotor speed more
slowly during the initial acceleration could possibly help avoid
convection.<br>
4. Make sure the cells perfectly aligned with the center of
rotation. I know that one rotor we have has inner and outer scribe
marks on each rotor whole and the two marks do not agree. I believe
that BITC sells a jig of some kind that aligns cells in the rotor more
reproducibly than the human eye using the scratches on the rotor as
guides.<br><br>
Further theoretical musings on convection and possible
prevention.<br><br>
If slower acceleration helps prevent convection, maybe an experiment
should be tried at few lower speeds also if convection is
suspected. By doing this you will sacrifice resolution, but may get
more repeatability. <br><br>
Also, I seem to remember that one reason the epon centerpieces have
aluminum or charcoal filling is to make them better temperature
conductors. I don't know which is better, but I would think the
aluminum filled would be better conductors of heat. So maybe using
aluminum centerpieces would help prevent convection not only be making
the original temperature equilibrium more effective but also by
dispersing the heat of initial compression more effectively. When
the centrifuge is started, the rotor expands and cools and the sample and
reference solution are compressed and heat up causing a small temperature
gradient. This acceleration caused temperature gradient may be a
cause of convection.<br><br>
There is the almost legendary story, which I have heard a few times from
a few sources, that Svedberg initially had rectangular cutout
centerpieces in his original centrifuge designs and then suddenly
realized during a trans-Atlantic trip that he needed the sector shaped
cells to avoid convection. This supports the advice above to have
clean, unwarped, perfectly aligned cells to avoid convection.
However, I wonder about the cleaning part: could the cell walls be
too clean? Since proteins adsorb to surfaces (which has actually
now been experimentally demonstrated by the fluorescence centrifuge
experiments), could a too clean wall actually cause a drag on the protein
solution near the wall and actually promote convection?<br><br>
I have also heard that the salt gradient that builds up in the centrifuge
actually stabilizes the sedimenting boundary. This is supported by
the mysterious action of fluid flow in the meniscus matching cells.
Even when the scribe line that lets buffer flow into the cell enters the
sample side at the bottom of the cell, the buffer solution tends to flow
into the cell and float to the top forming a laminar boundary. It
is amazing how smooth the flow and how sharp the boundary can be.
Similarly in synthetic boundary experiments, the lighter buffer usually
forms a single sharp boundary with the slightly heavier protein
solution. Here the buffer does come from the top side of the
protein solution. Convection here can result in a doubled synthetic
boundary that has more than one inflection point which becomes
obvious with diffusion if you collect enough scans. For
synthetic boundaries and the boundaries formed by meniscus matching
cells, convection occurs more with faster flow (the capillary is too
large or tilted or the rotor speed is raised too quickly).
<br><br>
I would conjecture then that conditions of low salt would tend to
destabilize boundaries and that convection would be more likely in these
conditions. In any case, even when you are constrained to examine a
protein in low salt conditions, it is a good idea to run a control
experiment of the same sample dialyzed into an almost identical buffer
with 100 mM salt added, hoping to get some indication if non-ideality if
affecting the sedimentation in the low salt conditions. I think
that irreversible aggregates would be unlikely to be dissolved merely by
adding salt, so this would help check reproducibility.<br><br>
It also seems that it may be worth trying to repeat an experiment at a
lower temperature if convection is suspected. More viscous liquids
tend to flow more smoothly and have less turbulence.<br><br>
And just by experience, it seems to me that I have most often seen
obvious convection at higher protein concentrations. It seems the
sharper the actually concentration boundary, the more likely it is to
convect. It is often the highest concentration cell that I am
throwing out of the data analysis even though it has the best signal to
noise ratio. Certainly, there is more fluid flow: both of the
solute and the back flow of the solvent with higher concentrations so it
makes sense to me that higher concentrations are similar to faster
sedimentation.<br><br>
<br><br>
This is what I have been able to come up with by asking a few people and
thinking about convection. I put the ideas out in public to see
what people think and to solicit other ideas.<br><br>
Cheers<br><br>
<br><br>
<br><br>
<x-sigsep><p></x-sigsep>
Dr. David B Hayes<br>
Boston Biomedical Research Institute<br>
64 Grove St.<br>
Watertown, MA 02472<br>
617-658-7738<br><br>
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