User:Sim Huay Ping/Notebook/CBE/08/150/2008/10/29

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Using the Applied Photophysics Chirascan Circular Dichroism Spectrophotometer to determine the secondary protein structure of the AMPs

The secondary structure of AMPs can consist of 3 kinds, the alpha helix, beta sheet or the random coil.

Starting up the instrument:

1) Turn on the Nitrogen supply tank and purge the system for 10 minutes. The levels should be as follows:

Lamp – 1 Monochromator -3 Sample -1

2) Turn on the “System” switch, the CD will respond and the status light should be stable and green. Let the system stabilize for 15-20 minutes prior to igniting the lamp.

3) Turn on the Lamp power, wait for 20 seconds then ignite the lamp by pressing the red Start button.

4) Turn on the water circulator followed by the temperature controller. Ensure that water is flowing through the tubes and that the water level is at the appropriate mark.

5) Open “Chiralscan” on the PC. The machine is ready to use when the Rx and Tx lights are blinking red.

Running basic samples:

6) Open the “Chiralscan” program. Some important settings include: The temperature - Go to “Settings” in the temperature section The range of the wavelengths you wish to analyse – After choosing, press “set” The step increments – the increment of the data collected The bandwidth – recommended 0.5nm The sampling time – time per each point taken, recommended 1 sec/0.5 nm

7) Put in blank sample (i.e. buffer only) and click on Baseline. This will measure a baseline which if you wish, you can auto subtract from subsequent samples ( or save this and manually subtract where needed).

8) Samples in the cuvettes should be bubble free. For slimmer cuvettes, spacers are available to ensure a proper fit in the cell chamber.

9) Before collecting the data, go to the “Spec0” icon in the tool bar to personalize the filenames.

10) To start the scan, press “start” near the bottom of the Chiralscan program. The data is displayed in Pro-Data Viewer. When the data begins collection, go to “Windows” and choose, “New Window”, select “HT” so that you can view the CD data (mdeg) as well as the HT voltage – DO NOT LET THE HT VOLTAGE GO ABOVE 800V.

Turning off the instrument:

11) Once you are finished, log out of the Chiralscan program. Turn off the lamp and the System switches and turn off the temperature controller and the waterbath. Allow the Nitrogen to purge for 10 minutes then turn off the Nitrogen supply.

12) If you book an afternoon or evening slot, please check to see if there is a user in the time slot immediately before you – If there is, please email them to tell them to leave the machine running (it is best if the machine is not turned off and then turned on again). If an hour or two will pass before the next user, please turn off the lamp but leave everything else running. If no one contacts you about leaving the CD on for the next session, please ensure you follow step 11.

Saving your data:

13) Once you have completed all your runs. Go to “My Computer” and move your data into your group’s data folder. Open “APLData Converter”. You can then drag your files and drop them into the converter window. This will put the data in .csv format which can then be opened in normal graphing programs.

USEFUL INFORMATION:

Cuvettes:

For routine analysis in the far-UV range (260-190 nm), use Hellma 1mm pathlength cuvettes (cat 110-QS). These are good for concentration ranges of roughly 10-50 uM. For samples that are lower in concentration (below 10uM) it is recommended to use a 1 cm pathlength cuvette. For samples of higher concentrations, cuvettes with smaller pathlengths are necessary. For near-UV analysis (above 260 nm), one needs higher protein concentrations (10-50 uM) but analysed in a 1cm cuvette (recommended cuvette, cat 115B-QS).

Cleaning Cuvettes:

We recommend using a 2% solution of Hellmanex II cleaner available from Hellma. Fill the cuvettes with a 2% solution and incubate at 50 degC for about 1 hour followed by rinsing with plenty of water followed by ethanol. Other necessary items: If you are performing temperature denaturations, the temperature probe must be secured into the cuvette. It is recommended that a layer of mineral oil be placed on the top of the sample, then insert the temperature probe (so as not to obscure the path of the beam) and fix it in place with Teflon tape. DO NOT USE PARAFILM, it will melt at higher temperatures and cause a mess!




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