[RASMB] Reproducibility in densitometry measurement

Wed Oct 1 02:32:06 PDT 2014

Dear John,

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.

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.

All the best - Mattia
At 11:40 AM 9/30/2014 -0700, John Sumida wrote:
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>
>Dear John,
>
>
>
>Thank you for your response and suggestions.  We will try to work at 
>higher protein concentration, though we are somewhat sample limited.
>
>
>
>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).
>
>
>
>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?
>
>
>
>Thanks again for your time in considering my question and your willingness 
>to share your knowledge on RASMB.
>
>
>
>Best regards,
>
>John Sumida
>
>University of Washington
>
>
>
>From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Philo
>Sent: Tuesday, September 30, 2014 10:12 AM
>To: 'John Sumida'; rasmb at list.rasmb.org
>Subject: Re: [RASMB] Reproducibility in densitometry measurement
>
>
>
>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.
>
>
>
>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.
>
>
>
>Remember that the protein samples will always behave differently with 
>regard to bubbles because the protein itself lowers the surface tension.
>
>
>
>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.
>
>
>
>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.
>
>
>
>John
>
>
>
>From: RASMB 
>[<mailto:rasmb-bounces at list.rasmb.org>mailto:rasmb-bounces at list.rasmb.org] 
>On Behalf Of John Sumida
>Sent: Tuesday, September 30, 2014 9:06 AM
>To: 'HGSR (Holger Martin Strauss)'; 
><mailto:rasmb at list.rasmb.org>rasmb at list.rasmb.org
>Subject: Re: [RASMB] Reproducibility in densitometry measurement
>
>
>
>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>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; <mailto:rasmb at list.rasmb.org>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>mailto:rasmb-bounces at list.rasmb.org] 
>On Behalf Of John Sumida
>Sent: 30 September 2014 00:50
>To: <mailto:rasmb at rasmb.org>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
>
>
>_______________________________________________
>RASMB mailing list
>RASMB at list.rasmb.org
>http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

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