F BICH 410 1st Edition Lecture 10 Outline of Last Lecture Protein Structure Protein Misfolding Outline of Current Lecture Primary structure determines tertiary structure so a minor change in primary sequence will cause undesired change in structure Protein Purification o Protein must first be purified before studied o First get protein out of cell through solubilization stabilization through temp pH buffer and must choose the best source of the protein Must first determine a method to detect protein of interest o Second use fractionation steps to separate protein of interest from all other o Visualize protein to confirm purity Protein purification must constantly ask how much total protein how much desired how pure Detection of proteins and amino acid o 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 specific activity o specific activity at max when no undesired protein Separation Techniques o Solubility salting in out Least soluble at pI o Salting in initially salts reduce electrostatic interact between proteins increase solubility o Salting out add enough salt and it will compete with proteins for interactions w water causing precipitation Fractionation use column chromatography o Consists of mobile phase stationary phase o Eluent solution added to column o Effluent what comes out of the column Gel filtration or size exclusion o Separated by molecular weight o 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 column o 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 affinity o Release from column by cleaving ligand from column or adding more ligand Visualaltion Techniques o 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 charge o 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 bonds o For heterodimer see 2 bands o For homodimer see 1 band
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