[RASMB] Questions on NONLIN

[lelj at interchange.ubc.ca]

Dear RASMB 'ers

First of all I would like to thanks all the people that replied to my email about the A to B association system. But yet I have many more questions!!! I realized that I need to better characterize my self-associating system before I can go on doing experiments of the complex.
Here are a list of questions that I would like to ask you (I'm using NONLIN), some are more general about experimental conditions other are more specific. 
I would like to thank you all in advance for the patient that you'll have in reading and hopefully answering:

1. Is it true that for value of sigma lower than 1 it's harder to fit your data (I do have WAY more problems when sigma=0.7-0.01 than sigma>1)?
If this is the case should I just run experiments at higher speed (20K, 30K) and forget about the lower ones (8K-12K)?

2.Is it better to run experiments in the 6hole cuvette or in the 2 holes? Why should I choose one over the other?

3.If at the end of my experiment I over speed (56K) to measure the absorbance at the meniscus is it wise to fix the values of delta=abs meniscus and don't let the value to float at all? Is this always the case?

4. I generally design an experiment as follow:
3 concentrations (in the 6 hole cuvette) 3 different speeds and over speed at the end. Then I try to fit these 9 data set. Can I try to fit together data set that comes from different experiments? (i.e. runs in different days with different concentrations? (With the same buffer!!!))?

5.I find that at my third speed and sometimes at my second speed, some of the protein is pelletted at the bottom of the cell..... Can I live with it and still try a global fit?

6.I work with a self-associating system, which is NOT monomer-dimer or tetramer, but I believe is monomer-12mer. 
I managed to fit 3 data sets at 20K and low concentration (measuring abs= 230nm).
First of all:  I can only globally fit these 3 data sets and data sets at lower speed don’t fit with each other and single data fit give me values different from the one at 20K. 
Moreover, it looks like the binding is very tight lnK2=9 (with K2=12 and sigma= monomer).  I calculate Kdissociation in Molarity to be=5E-15 M using the following equation: Ka(M-1)=[K2(Abs-11)x(e monomer x 1.2cm)E11]/12 and considering Kd=1/Ka.  The square root of variance is 7E-3 and I have nicely dispersed deviation. 
Does it make any sense having such tight interaction and be able to detect it with AUC? 
I also tried to fit using sigma=12-mer but I have no fit and also sigma=dimer or tetramer or 6-mer in equilibrium with other species but I have no fit.

7. Sometimes if I set sigma=monomer and allow N2 to float I have values that are not integer number or are bigger than what I expect (N2=15.5). If then I fix the value of N2 to a little smaller system (let’s say 12 instead of 15.5) I have the same fit with the same deviation and square root. (12 is the smaller number I can use to have a good fit!)
Should I always try to fit for N2 as small as possible?
I also tried to fit monomer- # -12mer where #=dimer/tetramer examer/ free to float etc. and I have no results. The free float give me #=0.2
 
8. With such a big associating system, is it enough if I can manage to global fit 9 data set or do I need more? (so far I haven't been able to fit more than 3 conc. at one speed.....)
Can I stay away from high concentrations?

Thank you all for your immense patience... 
Best regards,
Barbara


--
Barbara Lelj Garolla Di Bard
Dr. Mauk's Lab
Dept. of Biochemistry and Molecular Biology
University of British Columbia
2146 Health Sciences Mall
Vancouver, B.C. V6T 1Z3
Phone: (604) 822-2526