[RASMB] Reproducibility in densitometry measurement

HGSR (Holger Martin Strauss) hgsr at novonordisk.com
Tue Sep 30 01:28:53 PDT 2014


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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