[RASMB] downward sloping baselines or "i no longer have much hair left to pull out"

[John Correia]

Jack
 
You can estimate S from running the ligand alone, and then run the protein + ligand against buffer - ligand and then fit for ligand + protein interaction making the ligand a component in the reaction - many software packages can deal with this.  We often can go to another wavelength to avoid seeing the ligand (does the ligand absorb at 250 nm for example), but its often a great idea to include information about the ligand binding, sedimentation, stoichiometry in the fit.
 
Hair loss is a natural act so make the most of it!
 
 
 
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Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS  39216
(601) 984-1522                                 
fax (601) 984-1501                             
email address: jcorreia at biochem.umsmed.edu     
homepage location: http://biochemistry.umc.edu/correia.html 
dept homepage location:    http://biochemistry.umc.edu/ 
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>>> On 9/17/2007 at 10:25 AM, in message <46EE9C57.8030801 at alcor.concordia.ca>, Jack Kornblatt <krnbltt at alcor.concordia.ca> wrote:
I have checked the RASMB archives and there does not seem to be anything on this particular subject.
The problem is not the XL-I; the problem is an overly complex sample and reference.

The sample contains a protein that absorbs at 290 nm as well as a ligand that absorbs at 290 nm.
The reference contains just the  ligand. Both are in matching buffers.

The first scan: The baseline absorbance starts below zero and becomes more negative at higher radius.
Scan 100: The sample has almost sedimented completely giving the baseline absorbance what appears to be a "bow". 
Scan 200: The sample has sedimented completely and the absorbance is well below zero. It is more negative at high radius than low radius.

The problem is clearly the sample. I have not run the "control" in which the sample compartment contains no protein but does contain ligand; reference compartment contains no ligand.

This problem must have come up in the past. Needless to say, all analyses are feasible but they are not likely to be correct. I'm at wits' end.

thanks for any help you can give. i am attaching an xlgraph of the three scans.
jack kornblatt
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