Protein studies

Protein studies

1. Cell rupture to extract proteins

   • Methods
     • Potter-Elvehjem homogenizer
       • Thick test tube w/ tight fitting teflon plunger that moves up and down
       • Cells broken open, strained to remove connective tissue and blood vessels
     • Grind tissue in blender w/ buffer
     • sonication
   • Differential centrifugation
     • Centrifuge homogenate at 600g for 10min
     • Pellet 1: nuclei and unbroken cells
     • Centrifuge supernatant 1 at 15000g for 5min
     • Pellet 2: mitochondria, lysosomes, and microbodies
     • Centrifuge supernatant 2 at 100000g for 60min
     • Pellet 3: ribosomes, ER, Golgi, and plasma membrane fragments

2. Purification

   • Salt fractionation
     • Purification based on overall charge, ionic strength, and polarity (differential solubility)
     • Ammonium sulfate
       • High molarity at saturation
       • Easily centrifuged
     • Salt ions attract water away from protein
       • Protein molecules interact with each other and aggregate
         • Protein precipitates in native conformation
         • Proteins with greater hydrophobicity precipitate first
         • Multiple salt fractionations can eliminate contaminating proteins
     • Total activity = amount of target protein
     • Specific activity = total activity / total protein
       • increases with each step

3. Column Chromatography

   • Size-exclusion/Gel filtration
     • Stationary phase: cross-linked gel particles that form pores for eluent to flow through
       • Larger particles move faster because they are excluded from gel pores
     • Agarose or acrylamide gel
   • Affinity Chromatography
     • Column with substance S covalently bound to support
     • P1 molecules bind to S
     • all others eluted
     • high concentration of S elutes P1
   • ion exchange chromatography
     • Cation exchanger: “-” charged resin to bind “+” molecules, cations elute last
     • Anion exchanger: “+” charged resin to bind “-” molecules, anions elute last
     • neutral molecules flow thru
     • High salt content causes charge displacement --> protein elution
   • Reverse phase HPLC: nonpolar stationary phase, polar mobile phase
     • forward and reverse HPLC has fast elution time

4. Primary structure determination

   • Cleavage into Peptides
     • HPLC: Quantitative (amount of each AA) and qualitative (type of AA) analysis
     • Proteolysis
       • Trypsin--targets C-terminal side of basic residues
       • Chymotrypsin--bonds next to aromatic residues on C side
       • Cyanogen bromide--internal methionine residues
   • Edman Sequencing
     • Steps
       • Reaction with PITC
       • Formation of cyclic structure
       • Hydrolysis of peptide bond by anhydrous TFA
       • detemine identity of derivative

5. Electrophoresis

   • SDS-PAGE
     • Separates proteins based on size
       • smaller is faster--charge and shape usually similar
     • SDS=detergent to coat proteins with negative charge
   • Isoelectric focusing
     • Protein charge changes while moving thru gel
     • Migration under influence of electric field
     • Equal number of + and - charges
     • Stops when pH=pI (net charge = )
   • 2D electrophoresis
     • Isoelectric focusing and SDS-PAGE
     • improved separation of complex mixtures

6. Detection

   • mass spec
   • ELISA
   • western blot
   • ChIP