CD spectrometer protocol
- General protocol for turning on and off
- Wavelength scan
- Temperature melt :
- Chemical Denaturants:
- Sample preparation
- Commonly Recurring Annoying Problems:
- Turn N2 on for 20 min before turning the lamp on. Check that the pressure is high enough and adjust it between 4.5 and 5 using the knob behind the O2 detector.
- Turn on the lamp:
- INSTRUMENT POWER switch should be turned OFF.
- Turn ON the XENON LAMP POWER switch.
- Wait for the LAMP READY light to come on.
- Push the red IGNITE LAMP button.
- Note: the lamp will not turn on if the O2 concentration is higher than 7 ppm. Wait until it goes below 7 ppm before igniting the lamp.
- Note the lamp hours on log book, as well as your name, the date, etc.
- Turn ON the INSTRUMENT POWER switch.
- Start the computer if it is not already on.
- Start the Thermocube chiller, if it did not start together with the instrument (Power switch is on teh left side of the chiller).
- start the instrument DATA COLLECTION software (see note on troubleshooting if there’s a problem).
- Set up the experiment with Configure the Experiment.
- Select the datasets in Save Data Sets.
- Select the Data Path in the Displays: Data Browser.
- Run Experiments
- Perform averaging and math operations as needed.
- Save data via Data Browser
- Save any remaining data.
- Choose from the File menu "Terminate CDS Program" to shut down the software.
- The wavelength will move to the hom eposition, causing a delay in the program's termination.
- Note the lamp hours on the log book.
- Turn OFF the XENON LAMP POWER switch.
- Wait 10 minutes before turning off the nitrogen flow. Do NOT forget to do this!
- Clean cuvettes, syringe pump and other accessories if they were used.
- Turn OFF the INSTRUMENT POWER switch.
- Turn the CD on as described previously
- Select “Wavelength” as the experiment type on the CD Star program. This is the default.
- Set the start and end wavelengths
(default is 260 to 200 nm)
- Set the number of scans
(default is 1)
- Set the averaging time; the time the CD signal is averaged for a particular wavelength (default is 5 sec)
- Set the temperature
(default is 22oC)
- Set the data path
(default goes to the desktop on the Mac)
- Start the run. Parameters to note : dynode voltage (dynV) and CD signal (mo). 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.
- A good test for the instrument:
- run a wavelength scan of just air (w/o the cuvet).
- run a wavelength scan of just the cuvet (w/o buffer)
- run a wavelength scan of the cuvet and buffer.
- run a wavelength scan of the sample. Subtract the buffer signal.
- Wavelength data can be analyzed in Kaleidagraph, Excel or some other spreadsheet program
Purpose of wavelength scan:
- Find out whether your protein is folded or not.
- Gives an idea of secondary structure (sometimes)
- Determine the wavelength to do melt experiments
- Determine start and end points in melt experiments (either temp or chemical)
CD Melt experiment
You could do either a temperature or a chemical denaturant melt automatically.
- start the instrument as described previously
- change experiment type to temperature.
- Set start and end termperatures
- Set wavelength of observation (for alpha helices : 220-222 nm).
- Set averaging time (the time the data will be collected for and averaged). Default is 5 sec.
- 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 30oC 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.
- Set data path.
- You could also do the reverse experiment (going from high T to low T). This will check for reversibility (and thus the equilibrium assumption).
- Ready to go
- start the instrument.
- 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.
- 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.
- 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.
- 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.
- 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.
- The number of data points/shots you want. From experience, 35 data points give a good fit to the melting equation (see that protocol).
- Wavelength of observation (should be determined in wavelength expt)
- 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?
- Set the data path to your folder
- Other parameters are set by default and you can start the expt now
- 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 -30mo will make your life much easier later when you’re fitting the data.
- 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.
- 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.
- 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.
- The CD Star program starts very slowly. Reboot computer and press Shift key.
- 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.
- 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.
- 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.
Last updated Tue Oct 21 15:45:18 2003