[RASMB] Speed of Interference Optics vs. Absorbance Optics

Lake N Paul lpaul at purdue.edu
Wed Apr 13 07:14:30 PDT 2011


Everyone,
Thanks for the insightful responses! 
Ewa,
I have ran a boatload of AUC samples and I only see it with membrane proteins. I am getting a SEC MALS this month and this will be the first test case. When I said heterogeneous, I mean the system is dynamic, e.g. it has monomers, dimers and tetramers.

Holger,
I usually check my radial calibration every other week or so. I will double check it again. I ran a sample (non-membrane protein) before this and the distributions for interference and absorbance lined up perfectly.

Borris,
I think you are right in this case. The detergent is not detected at 280 nm (DDM, but will show up in the interference). I think the optic properties are vastly different. I suspect this because during the same run I had a GFP fusion on the same protein, the absorbance and interference peaks shift accordingly. The "control" peak of DDM (~3.1 S) lines up well in both samples (interference). This leads me to believe it is a property of the interference optics and the detergent -bound protein and not a hardware/software problem. I have managed to assign the most dominant peak (besides the DDM micelles) and was able to calculate the ratio of bound detergent (1.11 g/g) and the MW within 7% (using a method that John Burgner taught me). Cross linking data also supports this result (Weight-wise).

Thanks again, this has been really informative and helpful!
Best,
Lake

----- Original Message -----
From: "Ewa Folta-Stogniew" <ef55 at email.med.yale.edu>
To: "Lake Paul" <lpaul at purdue.edu>, rasmb at rasmb-email.bbri.org
Sent: Wednesday, April 13, 2011 9:07:04 AM
Subject: Re: [RASMB] Speed of Interference Optics vs. Absorbance Optics

At 11:56 AM 4/12/2011, Lake Paul wrote: 


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. 
First time in general, or first time for a membrane protein? 

When you say "heterogenous" what exactly do you mean by that? 

I had never collected data by AUC, but I would guess that size exclusion chromatography with absorbance, refractometry and light scattering detection will shed light on the state of this sample. From triple detection in SEC-LS/UV/RI I had seen cases where refraction will not align with absorbance for mombrane protein complexes. 

Ewa 



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 J 
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/ 


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