Team:Macquarie Australia/Protocols/spectralanalysis


Spectral Analysis of the BL21 Protein Samples

The following protocol was followed for the spectral analysis of the 1C3C #3, 6 and 7; and 4KE1 and 4KE3 protein samples. As a reminder the 1C3C protein samples comprise the fully assembled D. radiodurans bacteriophytochrome while the 4KE protein samples comprise the A. tumefaciens bacteriophytochrome.


Biliverdin (5 uL) was combined with 700 uL of sample in 5 2mL Eppendorf tubes. In addition, a negative control was prepared consisting of another 5 tubes of 700 uL of sample without BV. Each of these were wrapped together in foil and incubated overnight at 4 C. Overnight incubation was necessary here due to the slow reversion of the BP back to the red (660 nm) light absorbing conformation. Following irradiation at 660 nm we expect to see a change in absorbance at 720 nm due to a conformational change in the protein to the far-red light absorbing form.

Spectral Analysis

Samples were irradiated (660 nm) for 15 minutes using an LED light under low ambient light conditions. Upon irradiation, single, 80 uL digests of each sample were transferred to a 384 well plate for analysis in a PHERAstar plate reader, over the range 350-1000 nm and at a resolution of 2 nm. The wells are shown below. We blanked the instrument using 80 uL of the 4KE3 sample.

Wells in plate:

1: 1C3C #3 Irradiated

2: 1C3C #6 Irradiated

3: 1C3C #7 Irradiation

4: 1C3C #3 Control

5: 1C3C #6 Control

6: 1C3C #7 Control

7: Blank (4KE3)

Data Processing and Analysis

Raw spectral data was exported directly from the PHERAstar Data Analysis Software to Excel. Absorption maxima were normalized against the absorption maximum and graphed, as seen in the results here.