FGF Sample Preparation for Biophysical Studies

(Dr. Jihun Lee, June 5 2007)

 

Isothermal Equilibrium Denaturation: by Fluorescence (Varian, Cary Eclipse)

 

1. Prepare samples according to the “Isothermal Equilibrium Denaturation” formula (below)

 

  • final concentration: 5 uM
  • total volume: 700 ul
  • increment of GuHCl: 0.1M
  • numbers of sample: twice of Cm (e.g. if Cm=1.0 M, prepare samples till 2.0 M GuHCl)

 

2. Equilibrate the samples overnight

 

3. Measurement

 

  • Cuvette: 1cm path-length fluorescence cuvette
  • Temperature: 298K
  • Excitation wavelength: 295 nm
  • Emission: scan from 300 nm to 500 nm
  • Triplicate scans should be collected and averaged, and buffer traces are subsequently subtracted from the averaged scans. All scans are integrated to quantify the total fluorescence as a function of denaturant concentration

 

4. Repeat the full data collection at least three times to calculate standard deviations of thermodynamic parameters

 

 

 

 

Isothermal Equilibrium Denaturation: by circular dichroism (Jasco or Aviv)

 

1. Prepare samples according to the “Isothermal Equilibrium Denaturation” formula (below)

 

  • final concentration: 20 uM
  • total volume: 450 ul
  • increment of GuHCl: 0.1M
  • numbers of sample: twice of Cm (e.g. if Cm=1.0 M, prepare samples till 2.0 M GuHCl)

 

2. Equilibrate the samples overnight

 

3. Measurement

 

  • Cuvette: 1mm path-length CD cuvette
  • Temperature: 298K
  • Scan from 235 nm to 210 nm
  • Triplicate scans should be collected and averaged, and buffer traces are subsequently subtracted from the averaged scans. Data smoothing is performed prior to buffer subtraction using a five-point Fourier transform filter. The denaturation process is monitored by observing the change in CD signal at 227 nm with increasing GuHCl.

 

4. Repeat the full data collection at least three times to calculate standard deviations of thermodynamic parameters

 

Unfolding kinetics: by fluorescence (Varian, Cary Eclipse)

 

 1. Prepare samples according to the “Unfolding kinetics” formula (below)

 

  • Protein concentration: 25 uM
  • total volume of protein: 0.8 mL  (2.4 mL for total all three data set)
  • volume of buffer containing GuHCl: 0.9 mL
  • increment of GuHCl: 0.5M
  • numbers of sample: start from higher GuHCl than Cm à total 7 data points

      (e.g. if Cm=1.0 M, prepare buffer from 1.5M GuHCl to 4.5M GuHCl)

 

2. Equilibrate the samples overnight

 

3. Measurement

 

  • Cuvette: 1cm path-length fluorescence cuvette
  • Temperature: 298K
  • Excitation wavelength: 295 nm
  • Data collection times: 5 min to 20 min depending on unfolding rate

      (e.g. 1.5M GuHCl: 20min, 4.5M GuHCl: 5min)

  • Initiation of unfolding: mix 100 uL of protein with 900 uL of buffer à vortex à put the mixed solution in the cuvette à start measurement
  • Total manual mixing time: should be within 10sec

 

4. Repeat the full data collection at least three times to get the average rate constants

 

 

 

 

© 2007 Blaber lab