[RASMB] Reproducibility in densitometry measurement

John Sumida jpsumida at u.washington.edu
Mon Sep 29 15:49:32 PDT 2014


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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