FBICH 410 1st Edition Lecture 10Outline of Last Lecture - Protein Structure- Protein MisfoldingOutline of Current Lecture - Primary structure determines tertiary structure so a minor change in primary sequence will cause undesired change in structure- Protein Purificationo Protein must first be purified before studiedo First- get protein out of cell through solubilization/stabilization through temp, pH, buffer andmust choose the best source of the protein Must first determine a method to detect protein of interesto Second- use fractionation steps to separate protein of interest from all othero Visualize protein to confirm purity- Protein purification- must constantly ask- how much total protein, how much desired, how pure?- Detection of proteins and amino acido Through colormetry (coomassie blue), UV (most proteins have a Phe, Tyr, or Trp and absorbs at 280nm)- Desired protein detection can be made through enzyme assay and antibody binding assays- Purity= amount of desired protein/amount of total protein- If desired protein is an enzyme then amount is quantitated as activity. And purity is known as specificactivityo specific activity at max when no undesired protein - Separation Techniqueso Solubility- salting in/out Least soluble at pIo Salting in: initially salts reduce electrostatic interact between proteins increase solubilityo Salting out: add enough salt and it will compete with proteins for interactions w water causing precipitation- Fractionation- use column chromatographyo Consists of mobile phase, stationary phaseo Eluent- solution added to columno Effluent- what comes out of the column- Gel filtration or size exclusiono Separated by molecular weighto Largest protein comes out first and smallest These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Ion exchange- separate according to charge on a charged columno lastpositive protein sticks to negative column called a cationic exchange o Negative protein sticks to positive column called anionic exchange column How does protein come off column?- Through charge pH(careful of denaturing) or ionic strength o By adding a salt- anionic column add anionic salt- Ultracentrifugation- fractionate based on density and rate of sedimentary component- Affinity Chromatography- separate according to binding affinityo Release from column by cleaving ligand from column or adding more ligand- Visualaltion Techniqueso SDS Gel Electrophoresis- estimates molecular weight and purity SDS- sodium dodesce sulfate- detergent used to denature protein and disrupt subunit interaction All proteins coated with SDS have negative charge so separation based solely on size Large molecular weight found at top, small at bottom (travel farthest)o Native Gels- no SDS- separate by size and chargeo Compare migration of unknown to known standards to determine molecular weight Tells molecular weight of smallest unit- can’t determine subunit count SDS denatures proteins and breaks subunits apart- 1D Isoelectric focusing- determines proteins pI using 1D gel (has pH gradient)o Protein migrates to positive or negative until it reaches pH where proteins has no net charge and stops- 2D Isoelectric focusing- 1st do isoelectric 1D gel (pI) then apply to SDS gel (separates by molecular weight)o For Heterotrimer bonds will see 2 or 3 bondso For heterodimer see 2 bands o For homodimer see 1
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