Methods of Protein Analysis Flashcards
Amino Acid COmposition
Acid hydrolysis and amino acid analysis
- provides composition of protein expressed as the percentage of each amino acid
- little use for IDing proteins
Amino Acid Sequencing
Edman degradation: powerful method for protein ID
- provide sequence information at the N-terminus of a protein
- sequencing beginning on N-terminus is enough to identify a protein by comparison to genomic sequence data
useful for IDing post-translational modifications
Proteolysis
cleaving proteins into smaller peptides via specific proteases
trypsin
cleaves on the C-terminal side of basic residues
chymotrypsin
cleave on the C-terminal side of aromatic residues
Electrophoresis
movement of charged particles in an electric field; separates different molecules
- an ox (+); red cat (-); positive move toward cathode
- species with greatest charge move the furthest
- can be 1D or 2D
Immunoblotting
Western Blotting
- selective detection of proteins using antibodies
Mass Spec
determines the precise mass of molecules
- unique identifying information
Paper Electrophoresis
small molecules separated by electrophoresis on paper
- charge of species determined by pH of solution
Gel Electrophoresis (SDS-PAGE)
gel matrix forms channels through which proteins migrate; speed of migration dependent on size and charge
- SDS denatures proteins and measures based on mass (large proteins stay near top of gel)
Isoelectric focusing gel electrophoresis
separate proteins based on their isoelectric point
- charged proteins migrate until reach where pH=pI; net charge of zero=stop migration
Two-dimensional gel electrophoresis
isoelectric focusing and SDS gel electrophoresis
- powerful method for identifying a missing protein in tissue from a patient
X-ray crystallography and NMR
determine 3D structures; large quantities required
X-ray crystallography
structure determined via diffraction pattern
NMR
structures measured as chemical shifts
