6. The nitrogen generator is always turned on to supply the CD with a pure purge gas. If the nitrogen generator is not used the nitrogen cylinder is at least >500 psi (which is enough to last ~2 hours) (In the future if gaseous is supplied from a liquid nitrogen then the Dewar must be at least 15% full). 7. Turned on the nitrogen, gas valve behind the spectrometer, and record the time. 8. Set the turn on the computer timer for 5 minutes and remember if the chamber is not swept with nitrogen before the lamp is turned on the lamp life will decrease and the optics will become oxidized. 9. The nitrogen flow rate is at a minimum of 20 ft3/hr. The rate is measured at the top of the ball
10. After 5 minutes turn on the spectropolarimeter. The computer is normally not turned off.
11. For temperature control, turn the cooling water on and the Peltier unit shown at the right.
12. CD spectra are collected and the data is stored either on the user's diskette, on the computer hard drive, or printed out (Data on the hard drive may be deleted after 6 months). 13. Turn off spectropolarimeter, Peltier unit, water, and quit spectral manager program. 14. Set timer for 5 minutes. 15. Record any problems: spills, cuvettes are clean, and all is in working order except as noted: 16. After 5 minutes, turn off nitrogen gas at (record the time)
Index of Procedures
A. Before Starting B. Sample Preparation C. Starting up D. Collecting data E. Parameters F. Shutting down G. Analyzing data H. Emergency Procedures I. Official Procedures J. Miscellaneous Notes
A. Before Starting
Sign up for time!
Decide what type of cell will be needed.
Design the experiment(s). Near UV to look at the tertiary structure of the protein (340-250 nm), or far-UV (250-180 nm) to look at the secondary structure of the protein. Sample: typically 0.5 mg/mL protein in a low salt buffer. Volume: 200 uL for far-UV, 2 mL for near-UV
Store CD data on a 3.5" diskette, CD/W, or make a directory to storing data on the instrument to be transferred via FTP, to any other computer. The JASCO software to process data can be downloaded from the installation CD.
New users see Keith for details on using the spectropolarimeter.
B. Sample Preparation
Samples should be filtered or centrifuges at 15K before adding to cuvette (and ideally de-gassed).
A good starting concentration is 0.5 mg/mL. With the "standard" 0.1 cm (i.e., 1 mm) cuvette, data can be collected down to 195 nm. The volume of this cell is 300 uL. Greater than 100 uL is OK. The 0.1 cm path length is fine for seeing if the protein is structured, and for following denaturation by monitoring bands above 200 nm. To measure protein secondary structure accurately (i.e., down to 180 nm), use the 0.01 cm "sandwich" cuvette. It's volume is about 30 uL. Probably average 4 or more scans, or increase the protein concentration to about 5 mg/mL. For the near-UV region (340-250 nm), use the 1 cm cell.
There's no concern for near-UV, remember 10 mM TRIS, Pipes, Hepes, Mes, Cacodylate, or MOPS cause problems at 200 nm, and even 10 mM NaCl is a problem below 190 nm in a 0.1 cm cell. Usually, the lower the salt the better. The best buffers are borate (pKa is 9) or phosphate (pKa of 7). NaF or KF is much better than NaCl. Even water can be a problem near 180 nm (hence the use of short path lengths). See chart for buffer and solvent cutoff.
C. Starting up
1. Fill in the checklist. This is required for safety and for billing purposes. The most important thing to remember is that the lamp when on must have nitrogen gas flowing over it. (See Emergency Procedure 1 for more details).
2. Turn on nitrogen flow (i.e. turn the knob on the "gas" outlet from the nitrogen cylinder (or liquid nitrogen tank) counter-clockwise). The regulator, which is set, should read 19 psi.). The flow meter, on the far left hand side of the spectropolarimeter (the "spec"), should read 20 cubic feet per hour using the top of the indicator ball (20 ft3/h = 9.4 L/ min.). If not see procedure C (see part I).
3. After 5 min., turn on the spectrometer and computer. There must be no sample in the holder (otherwise there will be a "HT voltage" error).
4. If temperature control is needed, turn on the water to a slow trickle, and then turn on the Peltier unit (under the photo multiplier tube on the right). Press "start" on the Peltier. Check the flow of the water again after 10 minutes (it usually slows down).
5. Remove round cover and place sample in cuvette holder. The cell face is directed toward the photomultipler. A spacer may be required. The direction (and placement) does not make much of a difference, but be consistent. Always place the narrower cuvettes to the left of the spacer, with the numbers on the cuvette facing left. Note the hole in the space (about the diameter of a marker). This is what the light passes through. The solution in the cuvette has to cover this window completely and face the photo multiplier.
6. Replace the cover, shut lid firmly.
D. Collecting the data
1. Start the Spectra Manager Program from the icon on the desktop (Programs-->Jasco-->Spectra Manager). The PC computer in normally not turned off. The JASCO CD manual is pretty good. Please do not enter the Spectra Manager Setup section.
2. Double click on a desired program from the displayed menu. There are about 10 of them, including plain old spectrum measurement, and programs to measure spectra (either single wavelength, or full spectra) as a function of temperature.
3. Find the accessories, and select the Peltier unit and/or titrator if needed. Set the temperature using the computer.
4. Enter the parameters desired (Left most panel). See part 5 for selecting parameters. Enter "OK" rather than "Save".
5. Check and alter the "Options", which should be the far right panel. Enter the sample conditions, operator name, etc. Answer "OK" rather than "save".
6. The Data file is useful. Select Autosave, and the instrument will append a 00# to the selected file name selected.
7. Start spectrum measurement. The parameters can not be altered (even the options!) once this has started. The CD signal and HT channels are standard. The data is only reliable if the HT number is under 800. The HT can be converted to the absorbance in the spectrum analysis program.
Run a buffer check in the cell before using that cell for a protein. Precipitated proteins on the surface of the cell will look like beta-sheet! For that reason, consider buying a cuvette for CD measurements. In the absence of absorption, the HT voltage with buffer should be 260 ± 20. If not, make sure the lid is closed securely and the cell spacer is facing the right direction. If the HT voltage is very high the correct cell face may not be facing the pototmultiplier, i. e. no light is leaving the spectrometer.
8. On completion, the data automatically transfers to the spectrum analysis program. It's a good idea make sure the data is saved at this point. To export the data to another program (i.e., secondary structure deconvolution programs, save a copy of the data as "ASCII". Create a personal folder on the computer for the CD data and designate the folder as shared in order to transfer data from the instrument computer to a desk top computer.
E. Parameters
1. For far-UV, collect from 260 down to 200, 190, or 180 (depending on the sample and cell width). For near-UV, collect from 340 to 250. There are convenient parameter files for survey spectra (that take about 3 min.) and normal spectra (that take about 15 min.) for both near and far-UV.
2. Set up a quick scan, with a scanning speed of 50 nm/min. and a response time of 1 seconds. If the HT value is over 800, the data is suspect. If it does, either decrease the sample concentration or go to a shorter path length.
3. For a good scan in the far UV, use a scan speed of 5 nm/min., and at of 2 or 4 sec. Use 0.5 or 1 nm data point resolution and 1 or 2 accumulations. The secondary structure deconvolution programs usually need data starting at 260 nm. and down to 200 (more accurate if down to 180). For a good scan in the near UV, use a scan speed of 2 nm/min, at of 4 sec., a step size of 0.2 to 0.5 nm and 2 to 4 accumulations. These spectra will take 10 to 90 minutes to collect. Scan speed and time constant are interdependent, and their product should not exceed 0.33 nm (convert the time to minutes). Remember that a helical protein will have a stronger far-UV CD spectrum than a beta sheet protein, which will require more averaging.
1. Turn off the Peltier first then the water. By the way, if the Peltier is on without water, it make a noise, but probably not damage the unit.
2. Turn off the spectra manager program, and then the spectropolarimeter (green button). Turn on timer for five minutes. When the time has gone off, then turn off the nitrogen.
3. Complete checklist. Leave a copy of the checklist in the binder, and be sure to ask if there are any questions.
4. Cleaning the cells. In all cases, please rinse the cuvette with distilled water, then lab soap, and then water 5 times or so. If there is precipitated material, the fastest way to clean this is to add the Hellma cleaning solution. Alternatively use 30% hydrogen peroxide -- fill up half the cell, and then top it off with concentrated sulfuric acid. The cuvette will heat as the acid's added, and there is a small chance that the mixture will boil, so be sure to have protective wear on. Sometimes 10% NaOH will clean off material if the other does not work, it may etch the surface.
G. Analyzing the data
All the programs for determining secondary structure of proteins require export from the JASCO system as ASCII files. Even with the best data, the error in calculated helical content is 10%, the error in b sheet is much larger. The spectrum itself, however, is diagnostic for the folded form of the protein, and can be used phenomenologically.
The CDNN program, which is Windows 95 based and will read in a JASCO ASCII file only if the HT values are stripped. The HT values can be removed by using the spectral analysis program, finding "move channel", and selecting the CD data only. This will open only the CD data in a new window that can be saved as "ASCII". The CDNN program needs data between 210 and 260 nm and is reasonably self-explanatory. The web site access is: http://bioinformatik.de.
H. Emergency Procedures
1. Do not run out of nitrogen! The ozone made when there is no flow of nitrogen will fry lungs (and the mirrors and optics inside the spectropolarimeter thereby degrading the performance and interfering with the ability to get the data. Here is what to do. :
Quickly turn off the spectropolarimeter. Do it QUICKLY! Either a cylinder of gaseous nitrogen or liquid nitrogen set to release gaseous nitrogen can be used to run the spectrometer.
2. If there is a popping noise, or the smell smoke. Turn off the apparatus.
3. The machine suddenly quits. Check the circuit breaker.
4. Do not drop a $200 CD cell on the floor, shattering it. Get out the wallet.
I. When in doubt
Procedure A. Read the NMSU procedures on the Instrument Web pages.
Procedure B. Consult the Model J-810 Spectropolarimeter Operation Manual. It also sometimes refers to their older 450W lamp, which makes more demands than the modern 150W lamp.
Procedure C. Call Keith.
Procedure D. Read the two text books concerning CD. "Circular Dichroism: Principles and Applications" and/or "Organic Conformational Analysis and Stereochemistry from Circular Dichroism Spectroscopy".
J. Miscellaneous Notes
The cells work fine at 1/3 their capacity (half filled cells do not cause problems).
