Methods Of Protein Analysis

Question 1

Amino Acid Composition

Answer 1

• cleave covalent peptide bonds that link AA in PP chain via harsh chemical conditions to create individual AA residues
• separate AA via ion exchange chromatography to enable spectroscopic detection
• ID by elution profile

Question 2

Amino Acid Sequencing

Answer 2

• successive cycles of Edman degradation can provide sequence information for 15-50 residues at the N-terminus of a protein which is usually enough to ID a protein
• also helps ID post translational mods

Question 3

Proteolysis

Answer 3

Cleave protein into smaller peptides using proteases

• usually trypsin and chymotrypsin are used
• protein fragments are separated and analyzed via electrophoresis and mass spec

Question 4

Electrophoresis

Answer 4

Separate macromolecules such as peptides, proteins, DNA, RNA
Mixture is applies and subjected to electrical field
Species migrates from positive to negative pole based on size and charge

Question 5

Immunoblotting or Western blotting

Answer 5

Allows a selective detection of protein using antibodies that bind to specific antigen and then with secondary antibody that has fluorescent enzyme

Question 6

Mass spectrometry

Answer 6

Determine precise mass of molecule

Question 7

NMR

Answer 7

Used to determine structures of small proteins in solution via magnetic spin that aligns in magnetic field