Protein Isolation Flashcards
What are the steps in the Isolation of Proteins from tissue?
- Disrupt Tissue
- Release Target Protein
- Crude Fractionation
- Fine Purification
- Final Polishing
What are the TWO most common challenges in isolating proteins?
- Vast numbers of proteins
- Proteins may also contain other biomolecules
What are the steps for protein purification?
- Sedimentation & Fractionation
- Chromatography - scalable
- Electrophoresis - analytical
- Mass Spectrometry - confirms protein structure
What can induce the precipitation of proteins?
- Salts
- Organic Solvents
- Chaotropic Agents
- Change in pH of solution
What are the actions of Salting IN on proteins?
- LOW salt INCREASES solubility
- LOW salt PREVENTS aggregation and precipitation
What are the actions of Salting OUT on proteins?
- HIGH salt DECREASES solubility
- HIGH salt REMOVES solution sphere so they precipitate
What is a Chaotropic Agent?
A substance which disrupts structure of, and denatures, proteins
Which non-covalent forces do Chaotropic Agents interfere with?
- H-bonding
- Van der Waals forces
- Hydrophobic effects
What are FIVE common Chaotropic Agents?
- n-Butanol
- Ethanol
- Phenol
- Lithium Perchlolate
- Urea
Which interactions do Organic Solvents disrupt in proteins?
Hydrophobic
Which interactions do Salts disrupt in proteins?
Hydrophilic
How are precipitated proteins harvested after addition of Organic Solvent?
Centrifugation
What type of analysis are Organic Solvents used for?
Downstream analysis such as ELECTROPHORESIS
Which methods can be used to determine Protein Concentration during the purification process?
Absorbance / Fluorescence Spectroscopy
Which methods can be used to determine Specific Enzyme Activity during the purification process?
Enzyme Kinetics
