BL483 Biochemistry Techniques -- Lecture 1 -- Protein Assay
1. Introduction - Recalling Beer's Law
You should read the Introduction to Expt #1 and the Protocols in the text. We will not be doing the qualitative part of the expt - we will do only the quantitative photometric assays. Each student will do this lab by themselves. You will be given a numbered sample of lysozyme with an unknown concentration for you to use in this Expt. You will want to report the concentration of the protein in your unknown in mg/ml along with its number in your Lab Report for Expt #1.
A. Beer's Law Review
The basic principle underlying all quantitative photometric assays is
Beer's Law: A = Ëlc
- A= absorbance (usually at the wavelength of maximum absorption)
- Ë = Extinction Coefficient for the substance being analyzed (usually Greek epsilon)
- l = pathlength of the cuvette (usually 1 cm)
- c = Concentration (in units related to extinction coefficient, ie. 1/Ë)
If the Ë is a molar extinction coefficient (cm/M), the concentration is M or moles/liter.
The most useful part of Beer's Law is Absorbance is proportional to concentration.
When Beer's Law applies: A plot of absorbance versus concentration will be linear!
