Circular dichroism

We have access to circular dichroism at the PBCF for an hourly fee. You need to reserve the instrument in advance.

this paper is useful: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752288/

N2:

• Open main valve
• 50psi on left valve
• Need >500psi on right valve to maintain 50psi on left valve
• Close main valve when done w cd

On CD:

• switch system on
• switch lamp on
• turn on external temperature control
• turn on industrial chiller

Open ANMS software:

• Start lamp ignite sequence
• wait 20min for purge (bar graph should get to 1-3-1)
• green box on screen saying lamp is on
• on cd machine, yellowish headlight on left will turn on

Open chirascan

• Open pro data viewer
• Right click and set working directory to your folder
• In data viewer, preferences->always on line->local IP

In chirascan:

• leave absorbance box checked (which is the default)
• if running melt curve:
• open temp settings and set ramping temps (22 to 92C with 5C steps)
• set the T# probe that will be used, and place probe in cuvette
• in Advanced settings (Adv button), set the wavelengths to be monitored
• Best to look at spectra for that specific protein to determine the wavelength(s) to monitor
• generally, 222nm is best for helical proteins, 218 is best for sheety proteins
• run in Spectra-Kinetics mode

Spectra recommended settings:

• Low 200nm
• High 280nm
• Step 1
• (can go as low as 180nm and as high as 300nm, but hasn't been necessary for me)

Time per point: 0.1 to 1

• .25 recommended
• Lower number for weaker signal, but takes longer

Once cuvette loaded (buffer first), hit background

In spectra, below 220nm is mostly noise

• Solvents come out around 200nm

We don't have the CD analysis software, but I made a script to parse the resulting csv. In projects.andrew.helpers.parsers.parse_cd_data.py assuming you have saved the csv organized by wavelength, with temperatures are rows. Otherwise, use parse_transpose_cd_data.py. Graph with projects.andrew.visualization.plot_CD_meltcurve.py

Using 1mm cuvette which seems to be ok with 150ul sample

Cleaning the cuvette: hellmanex (just once), ethanol, water

Turning off: turn off light and pump in ANMS software close N2 shutdown CD system and light, then external temp control and chiller