[RASMB] SE interference data

mitrana at mail.utexas.edu mitrana at mail.utexas.edu
Thu Jun 26 17:31:53 PDT 2008


Hi Spinners

I thought I'd share my current situation and hear what the community has to say
-

1. Absorbance data at low protein concentration is almost as well described by a
monomer-dimer equilibrium model as a monomer-dimer-trimer model.

Monomer-dimer model: MW (fitted) ~ 69kDa, Ka = ~ 1.7 x 10^4 M-1.

Monomer-dimer-trimer model: MW (fitted) ~ 65KDa, K1 = ~ 0.5 x 10^4 M-1, K2 = 1.1
x 10^8 M-1.

Monomer MW (calculated) = 67kDa. Smack in the middle. The variance of the fit is
slightly better for the monomer-dimer-trimer model. So is the random-ness of the
residuals. The problem with the entire experiment is that the protein
concentration is not ideal ie it does not span the Kd value and is rather at
the lower end.

2. So I shifted to the interference system which allows a higher protein load.
Now sample preparation is a problem since I need to add a chemical to the
sample right before loading into the cell. So I cannot do the whole dialysis
business. The best I can do is try to add the same amount of chemical to both
the reference and the sample sectors.

3. I can fit the interference data with any degree of decency only to the
monomer-dimer-trimer model. The fitted values are slightly different from that
obtained from absorbance data. HOWEVER, I can do this only with data at low
rotor speeds ~ 13000 rpm. Higher speeds > 20K can't be fitted to anything - Not
even a fixed molecular weight model and you can usually fit any data to that
(BTW, fitting 13K rpm data to fixed MW model gives MW peaks at ~ 65-70 kDa and
~ 140-160 kDa). The fringes go upto ~ 25 which translates to the highest
protein concentration value of ~ 7.6 mg/ml.

Questions - 1. Is there any particular reason why higher speed interference data
can't be fitted? Might the high protein concentration have anything to do with
it - non-ideality or the steep gradient at high concentrations?
2. I am not very familiar with interference data. Is there any special treatment
for this type of data before analysis?

I'd appreciate any suggestions and advice during this dark hour of mine
(although I understand that it is summer and vacation time).

Mitra
-- 
Mitra S. Rana
Graduate Student
Institute for Cellular and Molecular Biology
2500 Speedway, UT-Austin
Austin, TX-78712



More information about the RASMB mailing list