Developing the Standard: PCB from Spirulina
We decided to use a standard used in many other experiments involving phytochromes. Phycocyanobilin (PCB) extracted from Spirulina is a commonly used standard, as Spirulina produces a large amount of chromophores.
Chromophores have a very high absorbance around 680nm. Here is an example of an absorbance spectrum from 300-800nm developed from the Quail Lab at UC-Berkeley.
We followed the protocol that the Quail Lab used to extract the PCB from Spirulina, and were able to produce the following spectrum:
The concentration is found by this formula:
We didn't trust the pathlength correction given to us by this spec so we ran the 1:100 and 1:1000 dilutions in the Stephanopolous lab, and here are the results that we got:
1:100 - A680 = 1.208
1:1000 - A680 = .425
After completing the calculation with the 1:1000 dilution, we got a concentration of 11.21372 mM, and we have a 4.5 mL solution.