Lecture 10: Application of protein isolation techniques in diagnosis

high levels of ALT and AST

liver damage

aspartate aminotransferase (AST)

converts oxalacetate → malate

more AST/ALT

fast decrease in absorbance

less AST/ALT

gradual decrease in absorbance

alanine aminotransferase (ALT)

converts pyruvate → D-lactate

specific activity

ratio of enzyme activity to the amount of protein in mixture

goal of purification

maximize specific activity

differential centrifugation

separates cellular components by size and density

whole cells, nuclei, cytoskeletons

low centrifuge force

mitochondria, lysosomes, peroxisomes

medium centrifuge force

microsomes, small vesicles

high centrifuge force

ribosomes, viruses, large macromolecules

very high speed centrifuge force

1. centrifugation

2. fractionation

steps of velocity sedimentation

velocity sedimentation

shallow sucrose gradient: 5-20%

equilibrium sedimentation

steep sucrose gradient: 20-70%

speed of sedimenting

velocity sedimentation separates by __

density of sediments

equilibrium sedimentation separates by __

salting out

effect by which most proteins are less soluble at high salt concentrations

large molecules

in dialysis, __ stay in bag

gel filtration

column chromatography that separates molecules by size, large molecules eluding first

ion-exchange

separates molecules by charge, opposites attract

affinity chromatography

separates molecules through ligand binding, major peak with non-binding proteins, second peak after eluting solution

HPLC (high-performance liquid chromatography)

separates molecules through absorbance, fine beads allow more interactions

gel electrophoresis

technique for separating proteins by size

SDS-Page

anionic detergent denatures proteins

2 amino acids

in SDS page, there is 1 SDS molecule per __

Coosmassie blue

in SDS-Page, protein separation is visualized by __

mass

in SDS-Page, mobility is directly proportional to __

lower bands

in SDS-Page, __ indicate more “normal” mass proteins are and travel faster

higher bands

in SDS-Page, __ indicate higher mass and travel slower

polyclonal antibodies

each antibody specific for one of the various epitopes

monoclonal antibodies

identical antibodies produced by clones of a single antibody-producing cell

monoclonal antibodies

antibodies used in a clinical research setting

hybridoma cells

monoclonal antibodies are produced from __

SS18-SSX

Synovial Sarcoma, driven by chromosomal translocation that produced __ oncogenic fusion protein

positive

a stain for SS18-SSX would indicate __ for synovial sarcoma diagnosis

ELISA (enzyme-linked immunosorbent assay)

plate-based assay that uses antibodies linked to an enzyme to quantify a specific protein in a sample

HRP (horseradish peroxidase)

enzyme that converts colorless substrate into a colored product in ELISA

indirect ELISA

type of ELISA with a goal is to quantify antibody levels

sandwich ELISA

type of ELISA with a goal to identify proteins of interest

prostate cancer screening (PSA)

involves protein produced by prostate gland that plays a role in fertility

elevate serum levels

result of PSA leaking into bloodstream

agarose beads

co-immunoprecipitation binding involves adding __ coated with an antibody-binding protein

Drosophila embryo

• stained with three different fluorescence-labeled monoclonal antibodies

• provides information on spatial organization of proteins

mass spectrometry

allows highly precise and sensitive detection of mass of an analyte by converting analyte into gas-phase ions and apply electrostatic potentials to measure mass-to-charge ratio (m/z)

fingerprinting

analyzing a trypsin-treated band in a gel-derived from a yeast nuclear pore sample