Help
Options
Didn't know it?
click below
click below
Knew it?
click below
click below
Don't Know
Remaining cards (0)
Know
retry
shuffle
restart
0:00
Embed Code - If you would like this activity on your web page, copy the script below and paste it into your web page.
Normal Size Small Size show me how
Normal Size Small Size show me how
Protein Analysis
Undergrad Biochem (Ch 5-d)
| Topic | Details |
|---|---|
| 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
Popular Biochemistry sets