[RASMB] Reproducibility in densitometry measurement

[John Sumida]

Dear Holger,

 

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.

 

What concentration range to you normally use for your measurments?

 

Thank you for taking the time to consider my question.

 

Best regards,

John Sumida.

 

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of HGSR (Holger
Martin Strauss)
Sent: Tuesday, September 30, 2014 1:29 AM
To: John Sumida; rasmb at list.rasmb.org
Subject: Re: [RASMB] Reproducibility in densitometry measurement

 

Dear John,

 

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:

 

-      It is an extremely sensitive instrument requiring a corresponding
experimental standard (which is higher than with a 4000 or 4500)

-      Make sure that the calibration is exactly as the measurement. For
example, don't leave the syringe in the plug under the
measurement/calibration. It will affect the resonator, at the 5th and 6th
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.

-      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?

-      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.

-      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.

-      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).

-      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)?

-      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.

-       

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.

 

Cheers, Holger

 

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: 30 September 2014 00:50
To: rasmb at rasmb.org
Subject: [RASMB] Reproducibility in densitometry measurement

 

Dear RASMB,

 

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.

 

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.

 

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.  

 

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.

 

My questions: 

Has anyone observed similar issues when performing this measurement?

Is there an optimal buffer system  for this measurement - perhaps at a salt
concentration less than 100mM?

Is there, perhaps, a subtlety in this measurement that I need to be aware
of?

 

Thank you for any and all comments returned.  They are greatly appreciated.

 

Best regards

John Sumida

University of Washington

 

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