Hi,
I am currently using Gdnhcl to analyze folding of my protein. I found the protein reach equilibrium at transition region(1.4M Gdnhcl) very fast(around 3mins). So I decide to use titration methods by adding 6M Gdnhcl to the protein samples. There is an increase in volume every time I add urea and it need a correction with volume change. This method was first introduced by Scholtz, J. M. But there is no more details about the correction. My first idea is to measure the correlation between the concentration of protein and CD value. It shows a linear relationship but there is always a difference in the coefficient. I also try to multiply the total volume by CD value. Is there anyone did that kind of correction? Thanks!
So what you are effectively doing is reducing the protein concentration. For each point and spectrum you need to calculate either the mean molar ellipticity or mean residue elliptisity with the new concentration. There is a very helpfull description of how to do this in Kelly, S. M. et al. Biochimica et Biophysica Acts (2005)
DOI: 10.1016/j.bbapap.2005.06.005
Hope this was of use
Hi Xinji
Correction for dilution should be enough, you can either look at the molar elipticity, but the CD change has two components in this case.
1) loss of signal because of the dilution. This one will be linear
2) The loss of signal also comes from unfolding of the secondary structure. This one will have a transition to be defined
Use raw data (mdeg) and, when you analize the transition, you will have a slope, which you can subract when you do the fittings to extract parameters. If you only want a phenomenological result, measure the slope of the line and subtract it from the rest of the points.
Having said all this, you should try to do it in separate tubes. From the consistencies you mention, it may not be as fast as you think. In fact, 3 minutes is too fast for a stable protein. Also, you often have transitions that are not observable by a signal change, but they do affect the equilibrium.
My suggestion: 1) use separate tubes, incubate 1h, and remeasure overnigth. If the curve is identical, then 1hr is enough to unfold. 2) use intrinsic fluorescence \, you can measure with the same sample as the CD.
If you really want to look at the stability and unfolding of your protein and extract parameters, you should have enough amount of protein and avoid the dilution.
If having enough protein is impossible, there is another option:
In separate eppendorf tubes, add (increasing) solid denaturant corresponding to the final concentration you want, taking on account the one you are carrying from the previous point. FIst point will be the zero denaturant. You remove the entire volume and add it to the solid of the next tube, dissolving the solid denaturant and spinnign down before measuring. Make sure you have enough buffer (you can check the pH of buffer you are using with the max denaturant concentration and see if it holds).
Gonzalo
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