Chapter 3: Protein Analysis Flashcards
Ion-Exchange Chromatography
Separates based on net protein charge
In cation exchange chromatography the beads have negative charge; hence the cations will be retained longer and anions will elute first
In anion exchange chromatography the beads have positive charge; hence anions will be retained longer and cations will elute first
Affinity Chromatography
Some proteins have high affinity for certain chemicals or functional groups
Beads are made with the specific chemical attached and the mixture is poured through column
Only proteins with affinity to the beads are retained in the stationary phase
Bound proteins can then be released
Assay
An investigative procedure used for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity
Gel-Filtration Chromatography
Mixture of proteins in a small volume is applied to a column filled with porous beads
Large proteins cannot enter the porous beads and thus elute first
Smaller proteins get difuse through the porous beads and are thus retained in the stationary phase longer
High-Performance Lquid Cromatography
Column has very fine particles to allow better separation by size
Smaller particles = more interaction sites = better resolving power
Requires application of pressure to obtain adequate flow rates
Often coupled with a detector to identify proteins as they elute
SDS−Page
Proteins are denatured and decorated with SDS which gives them a net negative charge
Proteins can be separated largely on the basis of mass
Small proteins move more rapidly through the gel and have greater relative motility
Larger proteins are more hindered and thus have a lower relative motility
Native−Page
NO denaturing agent is added in the preparation of gels; separation takes place on the basis of size AND charge
Conformation of the amino acid chains of the proteins are the factors that the separation is dependent upon
Proteins are not damaged in this process and can be recovered after the completion of separation.
Isoelectric Focusing
Isolates proteins by pI values by performing gel electrophoresis in a gel with an established pH gradient
Each protein will move until it reaches a position in the gel at which the pH is equal to its pI
Proteins differing by one net charge can be separated
Two-dimensional Electrophoresis
Isoelectric focusing is conmbined with SDS-Page to separate proteins on the basis of their pI and size
pH gradient gives pI separation while the polyacrylamide gel gives size and charge separation
Helpful for complex samples
Enzyme-Linked Immunosorbent Assay (ELISA)
Uses enzyme horseradish peroxidase or alkaline phosphatase to react with colorless substrate to create colored product
The enzyme is covalently linked to a specific antibody that recognizes a target antigen
If the antigen is present the antibody-enzyme complex will bind to it and on addition of substrate the enzyme will generate the colored product
Thus, presence of color indicates the presence of the antigen
Western Blotting
A polymer sheet is pressed againse an SDS gel which transfers resolved proteins to the sheet
Primary antibody is then added which detects desired protein
The antibody-antigen complex can then be detected by rinsing the sheet with a secondary antibody that is specific to the primary antibody
The secondary antibody is typically fused to an enzyme that produces a chemiluminescent or colored product
What bonds does trypsin cleave
Carboxyl side of lysine (K) and arginine (R)
What bonds does chymotrypsin cleave
Carboxyl side of aromatic amino acids (W,Y,F)
What amino acids does pepsin cleave
Aromatic residues (W,Y,F) with a preference for phenylalanine and leucine
What bonds do cyanogen bromide cleave
Carboxyl side of methionine residues
Edman Degradation
Chemical method used to determine the sequence of a peptide chain
Reads from the amino side of the protein
Amino Acid Synthesis
Block reactive amine side with tBoc
Activate carboxylic acid with DCC
α-terminal amino acid is anchored in a resin and C-terminal amino acids are added one at a time
Cryo-Electron Microscopy
Used to study large macromolecular complexes or proteins embedded within lipid membranes
