User:Klare Lazor/Notebook/Chem-496-001/2011/10/04

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Objective

Determine the fluorescence of MBP as a function of concentration. As well as, figure out the concentration of MBP based on its absorbance spectrum, and the sensitivity of the new fluorescence instrument. To determine the sensitivity of the fluorescence instrument, you will need to make a standard solution of Ru(bpy)(phen) in acteonitrile. Note: To get an accurate determination of fluorescence intensity, you don't measure just the intensity at the emission peak. You measure the integrated intensity of the entire emission.

Description

  • 0.2, 2.0, 10.0, 100.0 ), and 1000.0 ug/mL (stock solution) estimated concentrations of MBP were used. 266.05 µg/ml was the actual concentration of 1000.µg/ml.
  • Using the spectrum produced by the concentrations, the molar absorptivity was determine.
  • The real concentration of the estimated solutions were determined by using the equation of the line of the from the previous protien lab, y=.218x.
    • 0.2ug/ml--> .053211 ug/ml
    • 2ug/ml-->.53211ug/ml
    • 10ug/ml-->2.665ug/ml
    • 100.0ug/ml--> 26.6ug/ml

Data

Image:October_4.png

Notes

Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Thus, since a peak is seen a 280 nm we know the protein is concentrated enough at 100ug/ml or 26.6 ug/ml.


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