Molecular Methods: Proteins

Antibodies

Y shaped protein made by immune system to fight off antigens, bind epitopes on antigens

why is the antibody a y shape

one arm grabs germ other part signals to rest of immune system to destroy it

Fab region

antigen-binding sites, lock and key part shaped to fit only one specific target antigen

Fc region

bottom trunk of Y, acts as signal flare that immune system cells grab onto

epitope

The specific part or site on an antigen molecule that an antibody recognizes and binds to

antigen

molecule (often a foreign substance or pathogen) that the immune system recognizes as a target and can trigger an immune response

chromatography

laboratory technique used to separate the different components or parts of a mixture by passing mixture (mobile phase) through stationary phase material, sorting components based on size, weight, polarity

affinity chromatography

separates molecules based on highly specific biological interactions/chemical “lock-and-key” binding

ion exchange chromatography

separates proteins by utilizing differences in their net surface charge at a given pH, stationary phase (gel bead) carries a specific net charge, and proteins bind to it based on electrostatic interactions

Cation Exchange

Uses negatively charged gel beads to bind positively charged proteins.

Anion Exchange

Uses positively charged gel beads to bind negatively charged proteins

Size exclusion chromatography

laboratory technique used to separate molecules dissolved in a solution based on their size/molecular weight by packing column with tiny, porous polymer beads

electrophoresis

separates molecules according to charge and size by applying positive charge to bottom of gel, proteins drawn through gel

SDS-PAGE

common/powerful form of elecrophoresis, used in protein chemistry to analyze and determine purity/molecular weight of protein samples

SDS

unfolds/denatures protein

PAGE (polyacrylamide gel)

gel matrix where electric current applied, positive charge pulled towards bottom filtering molecules by mass

lower gel percentage

larger pore size, bad resolution, better for resolving alrge protein

high percentage gel

smaller pore size, better for resolving small proteins

gradient gels

best of both worlds

coomassie

blue color dye that binds to proteins non-specifically at basic Lys and Arg residues

western blots (immunoblots)

detects specific proteins

1. SDS-PAGE gel

2. transfer proteins to blot

3. block unoccupied regions of blot

4. add antibody against protein of interest (primary)

5. add labeled antibody against first antibody (secondary)

6. image the blot

western blot/immunoblot steps

primary antibody

binds to protein of interest in western blot, designed to recognize and lock onto specific epitope

secondary antibody

binds to all primary antibodies from a given species, can be mass-produced

fluorophore

chemical compound that absorbs light of a specific wavelength then re-emits it at a longer wavelength to identify secondary antibodies

1. coat plate with antigen

2. block

3. add primary antibody

4. wash

5. add enzyme-linked secondary antibody

6. add chromogenic substrate

7. measure absorbance

ELISA (enzyme linked immunosorbent assay)

1. coat plate with capture antigen

2. block

3. add sample to test

4. wash

5. add detection antibody

6. add enzyme-linked secondary Ab

7. add chromogenic substrate

8. measure absorbance

sandwich ELISA

• relies on competitive binding between radioactive and non-radioactive version of molecule

• measures radioactivity in found vs free fractions

• compare to standard curve to calculate amount of target substance present in blood

radioammunoassay

hematoxylin

stains acid nuclei blue

eosin

stains basic proteins in cytoplasm red/pink

PFA fixation

preserves tissues and cells

permeabilization

preparatory step for immunofluorescence/immunohistochemstry, uses detergent Triton-X, gently poke microscopic holes into cell membranes so that large molecules like antibodies can get inside

fluorophore-linked

antibody that has fluorescent dye (fluorophore) chemically attached to it