Experiment+3+-+Protein+Determination+by+Absorption+Spectroscopy

= Experiment 3 - Protein Determination by Absorption Spectroscopy =

Chapter 7: pages 213-232 ||= Absorption spectroscopy measures the light absorbed by a compound. The wavelengths at which a compound absorbs light depend on the nature of the absorbing molecules, giving that compound a characteristic absorption spectrum. The amount of light a solution absorbs depends on the concentration of the light absorbing solute and the distance of the light traveled through the solution. These observations are combined in the Beer-Lambert Law which states that absorbance increases linearly with solute concentration and with path length. ||
 * ~ Objectives of Experiment ||~ Reading in//Boyer// ||~ Principle of Experiment ||
 * = To learn methods for determination of protein concentration ||= Chapter 3: pages 69-83;

===Important Things to Remember from this lab: ===
 * **The Beer-Lambert Law**
 * [[image:http://imgey.com/images/eqn536yky.png width="800" height="145"]]
 * Absorbance is //unitless//. By definition, it is the ratio of light transmitted through the solution to the light entering the solution. A higher absorbance means that more light was absorbed by whatever solute is in solution
 * If this is the case, the units of E, C, and L must cancel.
 * [[image:http://imgey.com/images/eqn536hdh.png]]
 * A value for E is only valid for a particular substance //**at a single particular wavelength**//
 * Wavelength refers to the color of light passing through a solution.
 * [[image:http://imgey.com/images/304wavelen.jpg]]
 * Deviations from Beer's Law may exist due to:
 * Solution impurities
 * Instrumental imperfections
 * Operator induced errors (you being stupid)
 * Protein Spectroscopy
 * Proteins typically show high absorbance peaks at //**214nm**// (shown to the left of the enlarged area above) due to the peptide bonds absorbing at this wavelength.
 * Tyrosine (Tyr/Y), Tryptophan (Trp/W), and Phenylalanine (Phe/F) absorb at ~280nm due to their aromatic rings. Proteins with large amounts of these amino acids will produce high absorbance peaks near this level
 * In addition to the Beer-Lambert Law, there are two other methods for calculating protein concentration from absorbances:
 * Warburg-Christian Method
 * Depends on the relative absorbances at 280nm and 260nm
 * Gives a decent **estimate** of [protein] when 0.05<[protein]<2.0mg/mL
 * A280/A260 ratio will also estimate amount of nucleic acid (DNA+RNA) in solution
 * A solution with **NO** nucleic acids in it will have: [[image:http://imgey.com/images/eqn536fpf.png]]
 * Nucleic acid contamination will decrease this
 * Rule of thumb: [[image:http://imgey.com/images/eqn536zez.png]]
 * If nucleic acids are the only contaminants in solution, the protein concentration can be **esitmated** fairly well with the Groves Formula:
 * [[image:http://imgey.com/images/eqn536ksk.png]]
 * If other contaminants exists (in very small amounts) and [protein]<225ug/mL [protein] can be **estimated** fairly well with the Kalb-Bernlohr Formula:
 * [[image:http://imgey.com/images/eqn536aea.png]]
 * **//If a protein is available in pure form, its extinction coefficient can be determined and Beer's Law can be used to calculate its concentration. Beer's Law is the most ACCURATE method for calculating protein concentration if this is the case.//**
 * Know how to use the spectrophotometer!!
 * Blank before each use, at [protein]=0, Absorbance should = 0
 * When doing calculations, be sure to account for dilutions and/or sample size and/or aliquot size!
 * The typical volume of a cuvette is 3.00mL