CD Protocols

• General protocol for turning on and off
• Wavelength scan
• Melts
  • Temperature melt :
  • Chemical Denaturants:
• Sample preparation
• Commonly Recurring Annoying Problems:

General protocol for turning on and off

1. Turning on

1. turn N₂ on for 30 min. After about 20 min, turn water cooler on.
2. turn the power and lamp switch on (left switch). Wait until the lamp ready LED (green) is on, the press the “turn lamp on” button (RED button). You should see a bunch of red LED after the lamp is turned on.
   Note: sometimes the lamp doesn’t turn on, so you need to wait until the green LED shows up again BEFORE you press the RED button again.
3. Note the lamp hours on log book, as well as your name, the date, etc.
4. turn computer switch on (right switch)
5. answer “Y” to using CD with a cell holder and Mac CD software
6. start the CD Star software on the Mac (see note on troubleshooting if there’s a problem).
7. The CD is ready to go.

2. Turning off

1. quit CD Star software. This automatically quits the CD computer too
   ( you will see the word “Moving” on the right top side of the CD computer when it’s quitting).
2. note the lamp hours on the log book.
3. turn off lamp and CD computer.
4. turn off water cooler.
5. turn off N₂ after 5 min. Do NOT forget to do this or someone will give you a lot of grief.

Wavelength scan

1. Turn the CD on as described previously
2. Select “Wavelength” as the experiment type on the CD Star program. This is the default.
3. Set the start and end wavelengths
   (default is 260 to 200 nm)
4. Set the number of scans
   (default is 1)
5. Set the averaging time; the time the CD signal is averaged for a particular wavelength (default is 5 sec)
6. Set the temperature
   (default is 22^oC)
7. Set the data path
   (default goes to the desktop on the Mac)
8. Start the run. Parameters to note : dynode voltage (dynV) and CD signal (m^o). If the dynV is high, your noise is also high and the signal will be less accurate. DynV above 600 gives useless signal. Precision can be improved, as always, by doing multiple scans. DynV can be lowered by lowering protein concentrations or [salt]. Any additional components in your buffer can increase dynV.
9. A good test for the instrument:
   1. run a wavelength scan of just air (w/o the cuvet).
   2. run a wavelength scan of just the cuvet (w/o buffer)
   3. run a wavelength scan of the cuvet and buffer.
   4. run a wavelength scan of the sample. Subtract the buffer signal.
10. Wavelength data can be analyzed in Kaleidagraph, Excel or some other spreadsheet program

Purpose of wavelength scan:

1. Find out whether your protein is folded or not.
2. Gives an idea of secondary structure (sometimes)
3. Determine the wavelength to do melt experiments
4. Determine start and end points in melt experiments (either temp or chemical)

CD Melt experiment

Temperature melt :

1. start the instrument as described previously
2. change experiment type to temperature.
3. Set start and end termperatures
4. Set wavelength of observation (for alpha helices : 220-222 nm).
5. Set averaging time (the time the data will be collected for and averaged). Default is 5 sec.
6. Set mixing time (the equilibration time of the sample in the new condition). This variable is at the left bottom window, in the temperature box. For example, if the temp is increased to 30^oC and the equilibrium time is set to 30 sec, then the sample will mix for 30 sec before any CD measurement is taken. Default is 0.5 min.
7. Set data path.
8. You could also do the reverse experiment (going from high T to low T). This will check for reversibility (and thus the equilibrium assumption).
9. Ready to go

Chemical Denaturants:

1. start the instrument.
2. Connection to Mac. You need to restart the Mac and then press “Shift” until the screen displays a window. This is to connect the titrator to the Mac.
3. Start Star software. To activate the titrator to Mac, go to “Macros -> Communications Setup” and check the titrator box on the printer port. When you do this, watch the information window at the bottom of the screen. It should say “ Syringe pump responding correctly” or some such statement. This means the titrator can be controlled from the Mac now.
4. Prepare solutions of your protein in the native buffer and in the denatured buffer. Approximate volumes : native solution ~4 ml (2 expt) and denatured solution ~8 ml (1 expt). You should figure out the [Gu] you need to make from the wavelength expt.
5. Now set some parameter values in the experiment window. I assume you’re doing a GuHCl denaturation. Enter the value of [Gu] in your sample and denatured solution.
6. Enter the [Gu] to start and end the expt at. For example, you denatured protein is 6M, so you should end before that point, say 5.5M.
7. The number of data points/shots you want. From experience, 35 data points give a good fit to the melting equation (see that protocol).
8. Wavelength of observation (should be determined in wavelength expt)
9. Mixing and averaging times. Mixing refers to, well, mixing after the Gu has been injected into the cuvet. This needs to be long enough to get uniform mixing. I leave it to your own expert judgement on this time. Averaging time refers to how long the CD is collected before the data points are averaged. The longer the better, but if you have pretty clean data, why waste time?
10. Set the data path to your folder
11. Other parameters are set by default and you can start the expt now

Sample preparation

1. To get a good CD signal in the melt expt, make sure you experiment with various dilutions in the wavelength expt. Remember that you’re using a cuvet that’s 1cm in path length in the melt expt, while it is 0.1 cm in the wavelength expt. Your concentration should be changed correspondingly. A good native CD signal, i.e. around -30m^o will make your life much easier later when you’re fitting the data.
2. You can either filter or spin down your samples before the expt, if you’re really anal. Filtering with a 0.45µm filter would be enough to get rid of dirt and other crap.
3. Don’t forget to check [Gu] (or [Urea]) with the refractometer. If you don’t know how to use this, ask one of the friendly Baker lab members.

Commonly Recurring Annoying Problems (CRAP):

1. The temperature bars on the left side of the expt. window don’t show up. What’s going on? Turn on the temperature regulator (big machine right under the CD). To be distinguished from the water bath circulator.
2. The CD Star program starts very slowly. Reboot computer and press Shift key.
3. My data are so noisy, they’re keeping me awake at night! Normally this means that the lamp is old and not as bright as before. In this case, the dynode voltage increases and the signal becomes really noisy. As in the general case of noisy data, average, average and average.
4. My CD signal is kind of funky (scientifically speaking). Is this good? NO! Anytime you get a signal that’s going up and down, positive etc., i.e. abnormal behaviour in general, check the cuvet. Clean it thoroughly with EtOH, Hellmanex or water. Dry it completely too. Run the test expt outlined in the wavelength section of this protocol.
5. N₂ is running out. Can I keep using the instrument? NO! Notify the appropriate person in charge of N₂ ordering in the Baker lab. The day before your expt, you should always check the N₂ level to make sure you have enough.