<html>
<br>
Dear John,<br><br>
I fully second what Holger says about the concentration range. Apologies
if I'm going to say something trivial, but if total sample availability
is low, but you have enough for an initial point at relatively high
concentration (say around 2 mg/ml at least), then you can, by carefully
removing it from the U-tube and centrifuging it to remove potential
aggregates formed, prepare a second sample by dilution with the
equilibrated dialysis buffer, and so on.<br><br>
About determining the concentration, I agree with John Philo. If you know
the carbohydrate content with sufficiently high confidence (type &
number of any carbohydrate moiety), then you can use the PROMOLP software
we have developed (Spotorno et al., Eur. Biophys J. 25, 373-384, 1997) to
compute an extinction coefficient that will be way better than any RI
measurement (the calculation of dn/dc from composition is notorious for
being quite unreliable...). PROMOLP accepts as input the protein aa
sequence (either manually or from a file in the old swissprot format),
and then you can manually input all the carbohydrates types &
numbers. If you are interested, I can immediately send to you the Windows
version.<br><br>
All the best - Mattia<br>
At 11:40 AM 9/30/2014 -0700, John Sumida wrote:<br>
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Dear John,<br><br>
<br><br>
Thank you for your response and suggestions. We will try to work at
higher protein concentration, though we are somewhat sample
limited.<br><br>
<br><br>
Since you raise the question of knowing dn/dc and since the protein is
glycosylated, for us knowing the correct value for dn/dc is a problem. A
colleague of mine and I thought about comparing the interference
determination of the synthetic boundary with the absorbance signal
obtained in the same experiment. I have an extinction coefficient
determined for the sequence composition (as per Gill et al. Analytical
Biochem 1989) so we could determine the protein concentration (O.D. <
1.00). I could also assume that the protein portion of dn/dc =
0.187, and that portion corresponding to sugar was 0.146, (which I
understand to be the value for dextran). <br>
<br>
<br><br>
Since the interference measurement will be sensitive to glycosylation
whereas the absorption is not, I should be able to estimate a value for
dn/dc that better reflects the degree of glycosylation by determining the
relative contribution of the sugar component to dn/dc?<br><br>
<br><br>
Thanks again for your time in considering my question and your
willingness to share your knowledge on RASMB.<br><br>
<br><br>
Best regards,<br><br>
John Sumida<br><br>
University of Washington<br><br>
<br><br>
<b>From:</b> RASMB
[<a href="mailto:rasmb-bounces@list.rasmb.org" eudora="autourl">mailto:rasmb-bounces@list.rasmb.org</a>]
<b>On Behalf Of </b>John Philo<br>
<b>Sent:</b> Tuesday, September 30, 2014 10:12 AM<br>
<b>To:</b> 'John Sumida'; rasmb@list.rasmb.org<br>
<b>Subject:</b> Re: [RASMB] Reproducibility in densitometry
measurement<br><br>
<br><br>
John, it has been a long, long time since I last did any of these
measurements, so regrettably I don t remember any details, but I do
remember all too well that it takes quite a while to figure out how to
load the cell reproducibly without any bubbles, and that to get
reproducible results you really have to obsessive about doing everything
exactly the same way. <br><br>
<br><br>
If someone failed to clean your cell well and you ve got some junk stuck
on the surfaces that might explain your problems, so perhaps you should
try a more aggressive cleaning.<br><br>
<br><br>
Remember that the protein samples will always behave differently with
regard to bubbles because the protein itself lowers the surface
tension.<br><br>
<br><br>
But I definitely agree with Holger that you are using quite low protein
concentrations. Back in my Amgen days we would typically use
concentrations of at least several mg/mL, i.e. an order of magnitude
higher than you are attempting. <br><br>
<br><br>
Holger is also quite right that accuracy for the protein concentrations
is often a limiting factor. Determining the extinction coefficient via
the synthetic boundary approach assumes you know the correct refractive
increment, and that will vary significantly depending on the buffer
composition.<br><br>
<br><br>
John<br><br>
<br><br>
<b>From:</b> RASMB
[<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>]
<b>On Behalf Of </b>John Sumida<br>
<b>Sent:</b> Tuesday, September 30, 2014 9:06 AM<br>
<b>To:</b> 'HGSR (Holger Martin Strauss)';
<a href="mailto:rasmb@list.rasmb.org">rasmb@list.rasmb.org</a><br>
<b>Subject:</b> Re: [RASMB] Reproducibility in densitometry
measurement<br><br>
<br><br>
Dear Holger,<br><br>
<br><br>
Thank you for your detailed comments. Our experience with degassing
the sample is consistent with yours: does not appear to make much of a
difference. We have not tried removing the syringe during
measurement Anton Paar rep recommended that we leave it in the plug
during the sample measurement. We will try removing the syringe and
see if that helps.<br><br>
<br><br>
What concentration range to you normally use for your
measurments?<br><br>
<br><br>
Thank you for taking the time to consider my question.<br><br>
<br><br>
Best regards,<br><br>
John Sumida.<br><br>
<br><br>
<b>From:</b> RASMB
[<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>]
<b>On Behalf Of </b>HGSR (Holger Martin Strauss)<br>
<b>Sent:</b> Tuesday, September 30, 2014 1:29 AM<br>
<b>To:</b> John Sumida;
<a href="mailto:rasmb@list.rasmb.org">rasmb@list.rasmb.org</a><br>
<b>Subject:</b> Re: [RASMB] Reproducibility in densitometry
measurement<br><br>
<br><br>
Dear John,<br><br>
<br><br>
Your observations sound very familiar to our experiences with the DMA5000
and I would love to also hear from other users. Some comments from our
experiences:<br><br>
<br><br>
- It is an extremely sensitive instrument
requiring a corresponding experimental standard (which is higher than
with a 4000 or 4500)<br><br>
- Make sure that the calibration is
<i>exactly</i> as the measurement. For example, don t leave the syringe
in the plug under the measurement/calibration. It will affect the
resonator, at the 5<sup>th</sup> and 6<sup>th</sup> decimal place.
Likewise with the plugs or tubing at the other end. We fill our samples,
and then remove every tubing, plug, etc. That s also how we calibrate,
but others might have found a better way.<br><br>
- Gas bubbles can be a problem. Make sure
that the temperature of the samples and solvent is similar to your
measurement temperature before you dilute and fill them. Likewise, degass
preferably at the measurement temperature. We use the small vacuum pump
from our ITC-instrument, but in our hands, degassing did not make a huge
difference. You can also degass the solvent before you dilute the
protein, which can be done more thoroughly than with a protein in it. Any
other experiences out there?<br><br>
- 0.1-0.3 mg/mL is a rather low
concentration and small changes in the concentration due to adsorption of
your sample to surfaces (from syringes, etc) will have a relatively large
effect. But there might be a reason for the rather low
concentration.<br><br>
- Do you determine the density increment or
the vbar? A single solution/solvent pair is sufficient for vbar and the
highest concentration usually is the most precise measurement. You can of
course increase the precision of your parameter by repeated
measurements.<br><br>
- I m not aware that a particular salt or
concentration can have an effect but this is more testimony to my
ignorance (and bliss, for that matter).<br><br>
- Accuracy of the concentration
measurements is a huge issue and my impression is that the DMA5000 s
precision is better than the accuracy of most concentration measurements.
Are you using interference optics for the concentration determination?
Are you sure that the additional sample handling steps do not change your
concentration? Can you run N-determination or AA-analysis in parallel
(though they also have their issues)?<br><br>
- Finally, instruments from a certain
period have a CPU-board that will give out after a certain time (in our
case, after the warranty expired). I seem to be remember that our
measurements were less stable in the weeks before the instrument became
unusuable, but I have no documentation to this end. Check with your local
AntonPaar subsidiary.<br><br>
- <br><br>
There is of course the experimental alternative of using 18OH2, which
avoids the issue of concentration accuracy. But you will still need
accuracy in your density and s-values. Moreover, defining the solution
conditions wrt species concentrations becomes more of an issue.<br><br>
<br><br>
Cheers, Holger<br><br>
<br><br>
<b>From:</b> RASMB
[<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>]
<b>On Behalf Of </b>John Sumida<br>
<b>Sent:</b> 30 September 2014 00:50<br>
<b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a><br>
<b>Subject:</b> [RASMB] Reproducibility in densitometry
measurement<br><br>
<br><br>
Dear RASMB,<br><br>
<br><br>
We are trying to perform a measurement of v-bar using an Anton Paar
DMA5000. The measurement is of a glycosylated protein. The
densitometer was calibrated against a pure dI water standard and we ve
resorted to air checks between measurements to control for our cleaning
protocol: 3 x detergent wash, 3 x dI water rinse, 3 x ethanol rinse, and
air drying for 4 minutes or until the reading on the densitometer is
0.0012 g/cm3.<br><br>
<br><br>
We determined the extinction coefficient by synthetic boundary and
believe we have accurately measured the concentration of our
samples. Stock sample was dialyzed for a little over 48 hours
against PBS and dilutions of the stock into dialysate were made in order
to generate four solutions ranging from 0.1 to 0.3 mgs/mL.<br><br>
<br><br>
We are getting a values ranging between ~0.60 0.70, and are observing
that the density measurements are not reproducible beyond the fourth
decimal place. <br><br>
<br><br>
The reason for the lack of reproducibility appears to be that over time,
while the sample is in the U-tube, small gas bubbles are observed inside
the tube, either due to auto-hydrolysis of the solution or due to the
small vibration of the tube or something else I am not aware of. We
do not observe this for water alone. Samples were degassed, each
for 10 minutes.<br><br>
<br><br>
My questions: <br><br>
Has anyone observed similar issues when performing this
measurement?<br><br>
Is there an optimal buffer system for this measurement perhaps at a
salt concentration less than 100mM?<br><br>
Is there, perhaps, a subtlety in this measurement that I need to be aware
of?<br><br>
<br><br>
Thank you for any and all comments returned. They are greatly
appreciated.<br><br>
<br><br>
Best regards<br><br>
John Sumida<br><br>
University of Washington<br><br>
<br>
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