CD Checklist
Please fill out this form each time you're on the CD.
1. Print name
2. Date
3. Supervisorās name
4. Molecule name (optional)
5. Accounting information filled out for your lab
6. The nitrogen cylinder is at least >500 psi
(which is enough to last ~2 hours, or youāll have to switch tanks!)
(If using liquid nitrogen, the tank must be at least 15% full).
7. Turned on the nitrogen gas on at (print the time)
8. Set the timer for 5 minutes and read Emergency Procedure 1
9. The nitrogen flow rate is greater than 20 cf/hr
10. After 5 min., turn on the spectropolarimeter and the computer
11. For temperature control, turn water on and the Peltier unit
12. CD spectra are collected and the data is on diskette or printed out
(We will try to keep the data for 6 months).
13. Turn off spectropolarimeter, Peltier unit, water, and
quit spectral manager program.
14. Set timer for 5 minutes.
15. There are no spills, the cuvettes are clean, and allās in working order
except as noted:
16. After 5 minutes, turn off nitrogen gas at (print the time)
Basic Operation of the JASCO 810 CD Spectropolarimeter
Updated: Dec. 1, 2000
Table of Contents
A. Before you start *
B. Sample Preparation *
C. Starting up *
D. Collecting the data *
E. Parameters *
F. Shutting down *
G. Analyzing the data *
H. Emergency Procedures *
I. Official Procedures *
J. Miscellaneous Notes *
K. Acknowledgements *
Sign up for time!
Decide on your 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
A 3.5" diskette
See Jim or Betsey if youāre using the spectropolarimeter for the 1st time.
Samples should be filtered or spun at 15K before adding to cuvette (and ideally de-gassed).
A good starting concentration is 0.5 mg/mL. If you use the "standard" 0.1 cm (i.e., 1 mm) cuvette, you can collect data down to 195 nm. The volume of this cell is 300 uL. Greater than 100 uL is OK if you place the cell carefully. The 0.1 cm pathlength is fine for seeing if the protein is structured, and for following denaturation by monitoring bands above 200 nm. If you want to measure protein secondary structure accurately (i.e., down to 180 nm), use the 0.01 cm "sandwich" cuvette. Itās volume is about 30 uL. Youāll probably need to average 4 or more scans, unless you increase the protein concentration to about 5 mg/mL. If you want to look at the near-UV region (340-250 nm), use the 1 cm cell. Check the salt and buffer capability chart (see tables on next page).
Thereās no concern for near-UV, but that 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 pathlengths).
(see chart in Baleja lab for buffer interference)
5 mM imidazole is real bad
5% DMSO is bad, even as low at 240 nm.
Reference: Schmid, F. X., in Protein Structure, Chapter 11: Optical spectroscopy to characterize protein conformation, p. 269 (Creighton, T. E., ed.) Oxford University Press, 1997.
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 cf/h = 9.4 L/ min.). If it isnāt execute official procedure C (see part I).
3. After 5 min., turn on the spec and computer. There must be no sample in the holder (otherwise you get an "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 photomultiplier 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. You may need a spacer. The direction (and placement) doesnāt make much of a difference, but you should be consistent. I 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.
6. Replace the cover, shut lid firmly.
1. Start the Spectra Manager Program from the icon on the desktop (Programs-->Jasco-->Spectra Manager). If you donāt know your way around a PC computer, the 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. Thereās 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. Youāll want to check and alter the "Options", which should be the right most panel. Here you can enter the sample conditions, operator name, etc. Answer "OK" rather than "save".
6. The Data file is useful. You can select Autosave, and the machine will append a 00# to the file name you enter.
7. Start spectrum measurement. You cannot alter the parameters (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 (see part 8).
Run a buffer check first on the cell before using that cell for your protein. Precipitated proteins on the surface of the cell will look like b-sheet! For that reason, you may consider buying your own cuvette for CD measurements. See accounting information for more information.In the absence of absorption, the HT voltage with buffer should be 260±20. If not, make sure the lid is closed securely.
8. On completion, the data automatically transfers to the spectrum analysis
program. Itās a good idea make sure the data is saved at this point. If
you want to export the data to another program (i.e., secondary structure
deconvolution programs, you should save a copy of the data as "ascii".
You might want to create your own personal folder on the computer for the
CD data.
1. For far-UV, collect from 260 down to 200, 190, or 180 (depending on your 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 a t 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, a t 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.
Hereās a handy chart:
| Standard, Far | Standard, Near | Fast, Far | Fast, Near | |
| Start wavelength | 260 | 340 | 260 | 340 |
| End wavelength | 190 | 250 | 190 | 250 |
| Data Pitch | .5 | .2 | 1 | 1 |
| Scan Mode | Continuous | Continuous | Continuous | Continuous |
| Scan Speed (nm/min) | 10 | 5 | 50 | 20 |
| Response | 2 | 4 | 1 | 2 |
| Bandwidth | 1 | 1 | 1 | 1 |
| Accumulations | 1 | 2 | 1 | 1 |
4. In the temperature/wavelength scan program, be sure to check the
temperature sub-menu. It controls whether the sample is returned to the
original temperature. In this program, thereās no easy way to take a re-folding
curve. Perhaps can try the variable temperature program (which sits at
a single wavelength, rather than taking the whole spectrum)?
1. Turn off the Peltier first then the water. By the way, if the Peltier is on without water, it make a horrible noise, but probably not damage the unit.
2. Turn off the spectra manager program, and then the spectropolarimeter (green button). Turn on timer. When the timerās 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 you have 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 you have had precipitated
material, the fastest way to clean this is to add the Hellma cleaning solution.
Alternatively you can use 30% hydrogen peroxide -- fill up half the cell,
and then top it off with concentrated sulfuric acid. The cuvette will heat
as the acidās added, and thereās a small chance that the mixture will boil,
so be sure to have protective wear on. We keep a stock of the two cleaning
components in the right fume hood. Sometimes 10% NaOH will clean off material
if the other doesnāt work.
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.
My favorite is the CDNN program, which is Windows 95 based. This 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 is: http://bioinformatik.biochemtech.uni-halle.de/cdnn/index.html. I had no luck with the single installation file, but I successfully downloaded disks 1 and 2. Through the spectral analysis
1. You suddenly run out of nitrogen (Donāt let this happen ÷ ever!) The ozone you make when you run out of nitrogen will fry your lungs (as if we care, you worthless out-of-nitrogen lump), but more importantly it will fry the mirrors and optics inside the spectropolarimeter thereby degrading the performance and interfering with the ability to get the sacred data. This will also cost us big bucks to get the stuff replaced and at your salary you will be a very long time paying us back ... anyways, hereās what you do:
Quick like a bunny, turn off the specögreen switch, lower left. Just do it QUICK!
We can use either liquid nitrogen, or the boil-off from liquid nitrogen to run the spectrometer. If you want to finish collecting your data, youāll have to locate a spare. If not, usually nitrogen can be delivered the next day (or two). If you use up a tank, and donāt label it and tell someone itās empty, you will die a slow and painful death. Yes, one even more painful than having your lungs fried.
2. You hear a popping noise, and smell smoke.
Turn off the offending apparatus. Execute official procedures A, B, C, and D
3. The machine suddenly quits.
Check for popped breaker. Execute official procedures D, B, D again, and then C.
4. You drop a $200 CD cell on the floor, shattering it into a bazillion pieces.
Execute official procedures D and C, and get out wallet.
Procedure A. Get the hell out.
Procedure B. Consult the Model J-810 Spectropolarimeter Operation Manual. It probably wonāt do you any good. It also sometimes refers to a 450W lamp, which makes more demands than the modern 150W lamp we have.
Procedure C. Go get: Jim ( x6872 jbaleja@opal.tufts.edu)
Betsey ( x6873 bphili01@emerald.tufts.edu)
or Alan (x6873 alan@biba.med.tufts.edu)
Procedure D. Cuss like a sailor.
Rectangular cells: 1 cm, 3 mL
0.1 cm, 300 uL
0.01 cm, 30 uL
0.001 cm, 3 ? uL
0.2 cm, 600 uL (old cuvettes)
The cells appear to work fine at 1/3 their capacity (but 1/2 is easier to work with).
We have also circular cells, which apparently can give better quality data
than rectangular cells. We have a 0.1 mm pathlength, water-jacketed, 85 uL,
and a 1 mm pathlength, not water-jacketed, 313 uL. They canāt be used in the
Peltier unit, but we do the holder for them.
Sudha Veeraraghavanās (U. Texas, Houston) CD User Manual
John Osterhoutās (Rowland Institute, Cambridge, MA) CD User Manual
Current Protocols in Protein Science
Gosh, even the manuals that came with the Spectropolarimeter
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