purification, detection, and characterization of proteins

properties in which proteins vary

• size

• charge

• hydrophobicity

method of extracting proteins

break cell and remove cellular debris (membrane, DNA, RNA)

chromatography

separation of components based on their different interactions with an immobile (solid) material

mobile phase

liquid that moves past the solid immobile phase

movement of proteins in chromatography

rate depends on how much the protein interacts with the immobile material

gel filtration chromatography

• separates by size and shape

• solid phase consists of gel beads with small pores

• small proteins can enter and exit the pores, migrate slower

• large proteins cannot enter the pores, migrate faster

• proteins closer to being spherical migrate slower

ion-exchange chromatography

• separates by electric charge

• proteins with a net charge bind to the immobile phase with the opposite charge

• electrostatically-bound proteins can be released by flowing a salt solution through the column

antibody affinity chromatography

• antibodies specific for a protein are covalently coupled to the immobile phase

• all other proteins can be washed away, then the target protein can be released by lowering the pH

• bypasses all other properties of the protein

polyacrylamide gel electrophoresis (PAGE)

• protein mixture is loaded into wells of gel made from cross-linked polyacrylamide

• proteins migrate through pores

• smaller proteins migrate faster

• migration depends on size, shape, and charge

separation of molecules by electrophoresis

• direction of migration is determined by charge

• speed of migration is determined by net charge/mass ratio

SDS-PAGE

• sodium docecyl sulfate (SDS), anionic detergent

• SDS denatures proteins by interactions with its hydrophobic tail and the hydrophobic side chains of amino acids, disrupting oil drop structure

• polypeptide chain tends to be coated in a uniform layer of SDS molecules

• because SDS is negatively charged, protein structure is further disrupted

• all proteins migrate to positive pole

• all proteins are stained using non-specific dyes

• each band observed represents a different protein

immunoblot (western blot)

• proteins in SDS-PAGE gel are transferred to a membrane, exposing them to the surface

• primary antibody detects the protein of interest

• secondary antibody detects the primary antibody, used for detection through fluorescence or enzymatic activity

coomasie

blue dye used in SDS-PAGE

co-immunoprecipitation

use of an antibody that recognizes a specific protein to co-purify other proteins that interact with it

immunoprecipitate in co-immunoprecipitation

contains the protein carrying the epitope recognized by the antibody and any partner proteins stably associated with that protein

effect of GR-ligand in PPARa and GR interaction

• when GR-ligand is bound to GR, PPARa and GR are co-purified

• when GR-ligand is not bound to GR, PPARa is not co-purified with GR

• interaction between PPARa and GR depends on the presence of GR-ligand

immunofluorescence

• cells are fixed and immobilized on a microscope slide

• primary antibodies detect specific proteins

• fluorescently labelled secondary antibodies mark presence of primary antibody

fluorescence

property of certain substances to absorb light of a short wavelength and emit light of a longer wavelength

fluophores

electron in molecules that produce fluorescence

excitation

fluophores move to a higher energy orbital as a result of the absorption of a photon

emission

excited electrons release excess energy in the form of light when returning to their initial ground state

elements of fluorescence microscope

• light source capable of producing intense light over a broad spectrum

• filter cube

  • light from light source enters through excitation filter

  • light from fluorescent sample exits through emission filter

  • dichroic mirror: reflects one wavelength and transmits another

• objective lens

• sensitive camera

gene fusions

• generally done by cloning before introducing to the organism with a vector

• endogenous fusions can be done by homologous recombination

N-terminal fusion sequence

1. promoter

2. fluorescent gene (ex: GFP), for labelling

3. gene of interest

C-terminal fusion sequences

1. promoter

2. gene of interest

3. fluorescent gene (ex: GFP), for labelling

use of GFP in tracking

unlike immunofluorescence (where cells are dead), use of GFP can track cellular events in real time in vitro