Developing the Standard: PCB from Spirulina

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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.

QuailEx.png

We followed the protocol that the Quail Lab used to extract the PCB from Spirulina, and were able to produce the following spectrum:

1to100.png

The concentration is found by this formula:

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle C(mM)=\frac{A_{680}}{37.9}*\begin{cases} 100 (for 1:100) \\ 1000 (for 1:1000) \end{cases}}

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.