Lecture 2 protein Furci Flashcards
Slide 1 machine
ACCA - allows for specific protein purification despite time and difficulty. When protein needs to be de-bonded from organelle
Protein extraction
Biochemical investigations usually require pure components
Typical cell contains thousands of different substances
Many biomolecues have similar physical and chemical properties
Biomolecueles may be unstable and/or present in vanishingly small quantities
Purification of biomolecules is a formidable task
Would be considered unreasonable difficult by most synthetic chemist
Endotoxins
Can break down protein as we get to pure form.
Dimers, multimers, aggregates
Improper folding/bonding leads to this.
Lysozymes present
Can degrade protein of interest.
General purification strategy
Characteristics: Solubility, Ionic charge, Polarity, Molecular Size, Binding Specificity
Solubility (purification)
Salting in/out - adjust soln. to just below point of solubility for protein. Change ionic strength (add salt), change polarity (add organic solvent), change pH + temp can do this. Problem: precipiate other proteins. Separate soluble and insoluble material by centrifugation/filtration. Usually step 1.
Ionic Charge
Ion exchange chromatography, electrophoresis, isoelectric focusing
Polarity
Adsorption chromatography, Paper chromatography, reverse phase chromatography, hydrophobic interaction chromatography
Molecular size
Dialysis and ultrafiltration, gel electrophoresis, gel filtration/size exclusion chromatography
Binding specificity
Affinity chromatography
Salting in
Protien solubility increases with ionic strength, salt shields other protein charges (interactions). Basically allows for protein to be in functional state.
Salting out
Solubility decreases as you increase ionic strength.
Chromatography to purify
Size, Surface charge, Biorecognition (ligand specificity)
Size chromo
Size exclusion chrom - gel filtration
Surface charge chromo
Ion exchange chromo
Biorecognition chromo
Affinity chormatography.
Hydrophobic chromo
Exactly what is sounds like
Chromatography resins/matrices
Sepharose, agarose, dextran. Different ligands, rigidity, bead and pore sizes can be used.
Dailysis and ultrafiltration
Pore size for small molecules - stir bar pulls out smaller molecules, leaving larger ones inside. Intermediate step helping to get to purified product.