Sections
Protein Purification
Protein Extraction
Differential Centrifugation
Salting Out
Dialysis
Column Chromatography
Ion-Exchange Chromatography
Anion-Exchange Chromatography
Size Exclusion Chromatography
Affinity Chromatography
Specific Activity
HPLC
Spectrophotometry
Native Gel Electrophoresis
SDS-PAGE
SDS-PAGE Strategies
Isoelectric Focusing
2D-Electrophoresis
Diagonal Electrophoresis
Mass Spectrometry
Mass Spectrum
Tandem Mass Spectrometry
Peptide Mass Fingerprinting
Overview of Direct Protein Sequencing
Amino Acid Hydrolysis
FDNB
Chemical Cleavage of Bonds
Peptidases
Edman Degradation
Edman Degradation Sequenator and Sequencing Data Analysis
Edman Degradation Reaction Efficiency
Ordering Cleaved Fragments
Strategy for Ordering Cleaved Fragments
Indirect Protein Sequencing Via Geneomic Analyses

Concept #1: Spectrophotometry & Beer's Law

Practice: What is the relationship between light absorbance (A) & the amount of light transmitted through a sample?

Concept #2: Extinction Coefficient (ε)

Practice: Which of the following options is false for Beer’s Law?

Concept #3: Trp & Tyr Absorb 280 nm Wavelength of Light

Practice: A) Suppose myoglobin’s molecular weight is 17,800 g/mole and its extinction coefficient at 280 nm wavelength is 15,000 M-1 cm-1. What is the absorbance of a myoglobin solution (concentration = 1 mg/mL) across a 1-cm path?

Hint: Use Beer’s law.

a. 0.49            

b. 0.73            

c. 0.36            

d. 0.84


B) What is the percentage of the incident light that is transmitted through this solution?

a. 14%            

b. 6%

c. 21%

d. 58%

Practice: A protein solution has an absorbance of 0.1 at 280 nm with a path length of 1 cm. If the protein sequence includes 3 Trp residues but no other aromatic residues, what is the concentration of the protein? (Trp ε = 3,400 M-1 cm-1).

Practice: An unknown protein has been isolated in your laboratory and determined to have 172 amino acids but does not have tryptophan. You have been asked to determine the possible tyrosine content of this protein. You know from your study of this lesson that there is a relatively easy way to do this. You prepare a pure 50 μM solution of the protein, and you place it in a sample cell with a 1-cm path length, and you measure the absorbance of this sample at 280 nm in a UV-visible spectrophotometer. The absorbance of the solution is 0.398. How many tyrosine residues are there in this protein? (Tyr ε ≈ 1,000 M-1 cm-1 ).