FGF REFOLDING KINETICS MEASUREMENTS USING APPLIED PHOTOPHYSICS SX 20 STOPPED-FLOW

 

Dr. Jihun Lee

8/24/07

 

Sample preparation

 

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

 

  • Initial protein concentration: 5 ~ 20 uM
  • GuHCl concentration for protein denaturation: 2.5M

      (if Cm is higher than 2.5M, denature protein with 3.0M)

  • total volume of protein: 20 uL/ shot, require ~ 12 shots per each buffer concentration (6 shots for priming + 6 shots for data acquisition)

      à 20 uL x 12 shots x sample #

  • volume of buffer containing GuHCl: 4 mL (for duplication, prepare 8 mL)
  • increment of GuHCl: 0.05M
  • numbers of sample: start from 0.25 ~ 0.5 M GuHCl till nearby Cm

      (e.g. if Cm=1.0 M, prepare buffer from 0.25 M GuHCl to 0.9M GuHCl)

 

2. Equilibrate the denatured protein overnight

 

3. Measurement

 

  • initiate refolding of denatured protein by mixing protein with buffer 1:10 ratio
  • Temperature: 298K
  • Excitation wavelength: 295 nm
  • Emission wavelength: ~350 nm (ß use proper filter)
  • Data collection times: 5sec to 10 min depending on refolding rate

 

4. Usually repeat the full data collection twice to get the averaged rate constants

 

Click here to download the excel spreadsheet (see below)

 

To turn on the instrument

 

1. Turn on the “LAMP” switch

 

2. Ignite the lamp (push the red button)

 

3. Record the lamp hour in the log-in book

 

4. Open N2 gas à adjust the pressure (use the adjuster behind “sample handling unit”)

1:10 mixing (0.25 mL:2.5 mL) : 30 psi

 
 

 

 

 


5. Turn on the “SYSTEM” switch

 

6. Turn on the computer à log-in “SX users”

 

7. Open “Pro-Data SX” software

 

8. Set the working directory

1) Click “Pro-Data View” icon in the SX window

2) Specify the directory for data saving and analysis

    (C:\Documents and Settings\SX Users\DATA USERS\...)

3) Click “Set Working Directory”

 

9. Set the stop volume by screwing the “Drive volume adjuster” anti-clockwise

1:10 mixing à ~200 uL (4 turns)

 
 

 

 

 


10. Purge the system with NP-H2O

1) Ensure that the drive rams are pushed fully down

2) Set the drive valves to the “Load Position” (horizontal)

3) Fill up the “C” and “F” reservoir syringes with NP-H2O

4) Fill the drive syringes by pushing down the plungers of the reservoir syringes

5) Expel any air bubbles by flushing backwards and forwards between the drive syringes and the reservoir syringes several times

6) Fill the drive syringes with NP-H2O until both drive syringe pistons contact with drive ram

7) Set the drive valves to the “Drive Position” (vertical)

8) Empty any contents of the stop syringe by pulling the stop syringe piston upward at the “Empty Position” of stop valve

9) Change the stop valve from “Empty Position” to “Drive Position”

10) Push the NP-H2O from the drive syringe, through the flow circuit and into the stop syringe by pulling the drive ram upward

11) Repeat 1) ~ 10) steps 3~4 times

  

 

Figure 1. Sample handling unit (from Applied Photophysics SX manual)

 

 

 

Figure 2. Valve position (from Applied Photophysics SX manual)

 

Figure 3. Stop Valve position (from Applied Photophysics SX manual)

 

 

 

Sample loading

 

11. Purge the system with ADA buffer: procedures are same as step 10

 

12. Load buffer in the drive syringe C (“Load Position”)

1) First, fill the reservoir syringe with ~1mL of buffer and wash the drive syringe

2) Fill the reservoir syringe with ~3mL of buffer and load in the drive syringe C

 

13. Load denatured protein in the drive syringe F (“Load Position”)

1) Before loading the protein, wash the drive syringe with 2.5M GuHCl (ß same GuHCl concentration in which protein is denatured)

2) Denatured protein should be filtered and degassed (ß stirred for 10min under vacuum)

3) Load the protein in the drive syringe F

4) Make sure there is no air bubble

 

 

Program setting

 

14. Change the signal mode to “Fluorescence

 

15. Set the excitation wavelength to 295 nm

 

16. Set the detector voltages 400~500: has to be adjusted depending on protein signal. If the signal exhibits saturation, low the voltage until full range of intensity decrease is observed

 

17. Set the Trigger mode to “External”

 

18. Click “Seed” icon à type the file name

 

19. Set the “time (s)”

 

20. Set the number of data point (normally “1000” is recommended)

 

21. Set the numbers of scan by clicking “Repeat”

 

 

 

 

Data collection

 

22. Re-check both drive syringe pistons contact with drive ram

 

23. Change the valve position to “Drive Position” (vertical)

 

24. Prime the flow circuit and cell contents by clicking “Drive” at least 5 times

 

25. Click the “Acquire” to collect data

 

 

                     

 

 

Figure 3. ProData SX window (from Applied Photophysics SX manual)

To clean up the flow circuit and cell contents after data collection

 

1. Purge the system with 2.5M GuHCl

 

2. Purge the system with > 10mL of NP-H2O

 

2. Run the water-water scans (overlay of 5~6 scans)

 

3. Print the spectra and record “Date”, “Voltage”, and “Your name” à file in the SX20 log-in book

 

 

 

To turn off the instrument

 

1. Turn off the “LAMP” à Record the lamp hour in the log-in book

 

2. Terminate “Pro-Data Viewer” and “Pro-Data SX” window

 

3. Turn off the computer

 

4. Turn off the “SYSTEM”

 

5. Close the N2 gas

 

6. Release holding pressure using the adjuster behind “sample handling unit”