Chazin Lab Protocol for Determining Denaturant Concentration

notes

Written (2004-04-16)by Jonathan Sheehan

Adapted from Pace and Sholtz1

Purpose

When you really need to know the concentration of your urea or guanidinium HCl solutions,use this method.Especially GdmCl,because it's so hygroscopic,making weighing it out non- trivial.

Equipment/Materials

•You can borrow the refractometer fromlaura Mizoue

Measurement

1.Use 200μl of water or buffer to get your baseline

(should be near 1.333 at room temp.)

2.Use 200μl of your solution to get your unknown

(clean well,then return to Laura)

Calculate concentrations

1.[GdmCl] = 57.147 (deltaN)+ 38.68(deltaN)2 - 91.60(deltaN)3

2.[Urea] = 117.66 (deltaN)+ 29.753(deltaN)2 + 185.56(deltaN)3

3.where deltaN is the difference in refractive index between your solution and water

Note Solubilities (H₂0,25℃):

•Urea: 10.49 M

•GdmCl: 8.54 M

References

1.from:

Measuring the conformational stability of a protein by C.Nick Pace and J.Martin Scholtz in "Protein Structure: A Practical Approach" (Creighton,T.E.)pp.299-321,IRL Press,Oxford,1996.