I'm trying to compare the Rg of two similar small proteins (~7 KDa) using SAXS. Unfortunately, both proteins seem to show concentration dependence of their Rg where in one of them this effect is more robust. For both proteins at concentrations below 2.5 mg/ml the Rg increase is linear with concentration. What I am trying to do is to extrapolate these data to zero concentration and then use the Rg values derived from the Guinier plot of the extrapolated curves for comparison.
I'm a little bit puzzled with how to use the ZeroConc function in Primus. I load all my measurements of different protein concentration (buffer subtructed), modify the concentration field for each measurement and plot (fig 3).
Now if I hit "ZerConc" the multiplier field of each curve is modified and they no more overlay at high Qs as before (fig 4). The output curve is similar to the lower concentration curve. Its Guinier indicates a similar Rg to the lowest conc. but its I(0) is significantly higher. In analytical ultra-centrifugation studies using similar concentratios both proteins were shown to be monomers in solution. I therefore can't trust this curve since its I(0) reflects a molar mass much greater than that of a monomer.I also tried to set nBeg to 2 but got the same results
I saw these directions on one of the threads:
Please note, when you use "ZerConc", keep the following steps:
1) load data from the concentration seria and put the concentrations values
2) select the part of the curves where there is no the concentration effect and all curves coincide if they are scaled.
3) press "ZerConc" button, in the output you will get the curve where the
beginning part is extrapolated to zero concentration, "middle" part is the average from all curves and for higher angles part (if any) the data from the highest concentration are taken.
When I follow these directions (fig 5-6) the resultant curve and all the other do overlay at high Qs. Here the extrapolated curve's Rg and I(0) are lower than that of the smallest concentration curve but still, for one of my proteins using the reference I(0) of BSA or water, this I(0) reflcts a molar mass higher than expected for a monomer. In addition, in several cases following this second method I get an extrapolated curve as appear in figure 9 which cannot be used for analysis.
Which of the above is the rights way to go?
Is my way of trusting ZeroConc results only if Guinier gives an I(0) appropriate for a monomer right? how accurate should this mass calculation be?
Is there some way to get an output for the ZeroConc function to know the quality of its output?