[RASMB] Speed of Interference Optics vs. Absorbance Optics

HGSR (Holger Martin Strauss) hgsr at novonordisk.com
Wed Apr 13 04:00:39 PDT 2011


Hej Paul,

looking at your data, there are several observations:

- the meniscii from Abs and Int are not equal (ca. 5.97 cm for Int and ca. 5.93 cm for Abs). You might want to compare the radial calibrations from Abs and Int by scanning the CB. If you can't get them to overlap yourself, ask your field technician to do it. Distorted radial calibrations will give distorted data, will give distorted fits, etc.

- from the Int data, it looks as if you have at least three fractions present, two for the high s-region, one for the low s. The high s-range looks similar for Int and Abs (and your Abs-c(s) does indeed show two signals), so your Int c(s)-result seems too detailled for the amount of data/information in your sample. You might want to try a "simpler" approach like dc/dt or vHW.

- it looks as if the high s-region in your Int data is completely separarated from your low s-region. You could therefore try to determine the concentration of material in that region simply by taking the signal amplitude of the first plateau and extrapolate that to wexp2t=0. The same procedure for Abs would give you a handle on the signal of detergent co-sedimenting. From my experience however, it's a good idea to perform independent measurements of the single compounds for such kind of calculations.

Best, Holger

________________________________
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Lake Paul
Sent: Tuesday, April 12, 2011 5:57 PM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] Speed of Interference Optics vs. Absorbance Optics

Folks,
I have a sample, a membrane protein with 0.01% DDM. I collected data at 45000 rpm, 4 min/scan. The sample is very heterogeneous but I am getting a decent peak from the absorbance data (280 nm) at 7.4 S, but the interference data gives me a peak at 6.5 S. I am trying to calculate the amount of detergent bound so I need to align the interference data with absorbance data. This is the first time in which I have gotten the peaks from the interference data not lining up with absorbance data. Is this due to the time it takes to make an absorbance scan relative to the interference scans? And/or I just spun the sample too hard? Or is the DDM affecting the sedimentation? See the attached powerpoint.
Yes, the slit assembly is functioning properly :)
Any suggestions will be helpful!
Best,
Lake

PS I collected data during the same run with the same sample except it had GFP attached. The interference peak shifted accordingly. The DDM peak lined up well also.

Lake N. Paul, PhD
Biophysics Research Specialist
Discovery Park - Bindley Biosciences Center Purdue University
1203 West State Street
West Lafayette, Indiana, 47905
765.494.4960
BioAnalytical Lab Website:
http://www.purdue.edu/discoverypark/bioanalytical/

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20110413/d067c4a4/attachment.htm>


More information about the RASMB mailing list