Circular dichroism - glasgowlab/home GitHub Wiki

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