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Protein Analysis

Undergrad Biochem (Ch 5-d)

TopicDetails
Protein Analysis Steps 1. Extraction 2. Purification 3. AA Composition 4. AA Sequencing
1. Extraction Cell lysis ultracentrifugation
2. Purification (2 steps) a)Precipitation b)Purify
a) Extraction "salting out" protein dialysis to remove detergents and salt
b)Purification Column Chromatography Electrophoresis
Column Chromatography types i) Gel Filtration ii) Ion exchange iii) Affinity iiii) Hydrophobic
i) Gel Filtration aka size exclusion (SEC) - BY SIZE
ii) Ion Exchange based on charge difference between proteins
iii) Affinity use ligands bound to beads - selectively binds specific proteins
iiii) Hydrophobic aka Reverse Phase - based on hydrophobicity
Electrophoresis move molecules through polymerized gel within electric field - based on size, shape, charge
3. aa composition A. Hydrolysis B. Analysis
A. Hydrolysis (options) (a) soak in 6M HCl 10-100 hrs or (b) 6M HCl @110*C 18-36 hrs (destroys some aa)
B. Analysis (1) Edman degradation (2) (a) ion exchange chrom. (b) Reverse-phase HPLC (RP-HPLC)
Edman degradation Edman's reagent (PITC) modifies N-terminus of polypeptide
4. aa Sequencing (Steps) (1) cleave disulfide bonds < 50 aa (2) Cleave off ends repeatedly (i) Determine N-terminus (ii) Determine C-terminus > 50aa (4) Cleave into smaller pieces (5) Overlapping determined
(1) Disulfides cleaved by performic acid
(2) Determine N-terminal (options) (a) Sanger's method or (b) Edman degradation**
(a) Sanger's method treat w/ DNP derivative
(3) Determine C-terminus use pancreatic enzymes to cleave COOH end (a) Carboxypeptidase A or (b) Carboxypeptidase B
(a) Carboxypeptidase A cleaves aromatic residues
(b) Carboxypeptidase B cleaves basic residues
(4) Cleave into smaller pieces a) Trypsin b) Chymotrypsin c) Cyanogen bromide
a) Trypsin pancreatic enzyme most reliable cleaves on COOH side of Lys, Arg
b) Chymotrypsin pancreatic enzyme cleaves on COOH of Phe, Tyr, Trp
c) Cyanogen bromide cleaves on COOH side of Met
(5) Overlapping determined - study sequences fragments from each enzyme - determine original protein's overall sequence
Created by: kristin_leann
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