Protein Techniques

Bio 223 Exam 1

Purification

Extracting protein from intact organisms

Cell Culture

1. introduce double-stranded recombinant plasmid DNA to bacterial cell

2. cell culture products 100s of millions of new bacteria

3. extract protein

Homogenate

mixture of broken apart cell or tissue

• via sonicator, mechanical force, or lysis buffer

Centrifugation separates based on…

size/density

• supernatant & pellet

Chromatography

protein mixture is applied to a column that allows differential separation

• protein purification

• solvent flows through beads

Ion-exchange chromatography

based on charge

• charged beads

Gel-filtration chromatography

based on size

• porous beads

Affinity chromatography

based on binding

• beads with covalently attached substrate or antibodies → bind to enzyme molecules

Antibody

recognizes antigen/protein of interest

• different antibodies bind different proteins

• bind to pathogens

• extracellular space

Disulfide bond formation

Cysteine, oxidizing environment

• extracellular environment, can be interchain or intrachain

Immunoaffinity column chromatography

beads coated with anti-A antibodies

• elute pure antigen A

PAGE

polyacrylamide-gel electrophoresis

• porous gel, buffer, cathode, anode

• proteins travel through gels at different rates

• proteins run towards positive charge

• after gel, proteins are separated in “invisible” bands

Ladder

molecular weight standards for gel electrophoresis

Coomassie Blue

stains all proteins in gel electrophoresis

SDS-PAGE

gel electrophoresis with denatured (unfolded) proteins

• very common

• loss of secondary & tertiary structures

SDS

adds (-) charge

beta-mercaptoethanol

reducing agent

• breaks any disulfide bonds

Western Blots

SDS-PAGE to detect the presence of specific proteins

• label protein of interest with specific antibody

• will reveal protein of interest

Immunohistochemistry/immunostaining

antibodies can be used to determine which cells contain a protein

• couple specific antibodies to special tag (i.e. fluorescent dye)

X-ray crystallography & NMR spectroscopy

analyze protein specific 3D structure