Protein purification

Protein purification

1. Choose biological maaterial

2. Preparation

   • Obtain initial extract
   • Techniques
     • To breal the cells and get the proteins out.
     • dif. osmotic pressure
     • ultrasonic waves
     • detergents
     • organic solvents
     • french press
     • Homogenator
     • Blender
   • Differential centrifugation
     • Separate proteins from other nonsoluble particles based on difference of mass/density
     • supernatant with proteins

3. Purification

   • stabilize the protein
     • pH-neutral
     • T not exceed 60/65ºC
       • usually on ice4ºC
     • prevent air contact/foam, causes oxidation
     • protases-protease inhibitors
     • contaminants-antimicrobial agents
   • Methods
     • Solubility
       • Based on the precipirtation of proteins
       • Salting out
       • pI precipitation
       • precipitation with organic solvents
         • Act by making the solution less polar, they lower the dielectric cte of solutions There are less water molecules available to solvate proteins.
         • Ethanol
         • Acetone
     • Molecular weight
       • Dialysis
       • Ultracentrifugation
       • size exclution cromatography
       • Electrophoresis SDS gels
         • polyacrylamide sulphate catalyst
           • acrylmide
           • N,N-methylenebisacrylamide
     • Electric charge
       • Ion-exchange chromatography
       • Electrophoresis 2D
         • SDS electroph
         • Isoelectric focusing
       • Isoelectric focusing
         • ampholytes-gradient pH
     • Polarity
       • Adsortion chromatography
       • Reverse phase chromatography
         • liquid-liquid partition
         • protein is denatured
       • Hydrophobic interaction chromatography
         • hydrophilic stationary phase with hydrophobic groups covalently
           • native structure of protein is maintained
         • molecules bind by weak hydrophobic interactions
     • Specific binding
       • Affinity chromatography

4. Protein detection

   • ELISA
     • Enzyme-linked immunosrbent assay
   • Immunoblotting/Wetern blotting

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