Download
1 / 12
310 likes | 1.09k Views
Biochemistry Lab. Protein Purification - 1 Methods of Cell Lysis. Methods of Cell Lysis. Passive Methods: Freeze-thaw Chemical Enzymatic Osmotic Pressure. Physical Methods: Sonication Cell Bomb Homogenizer. Additional Considerations:. Volume Cell Type Toxicity Cost.
E N D
Biochemistry Lab Protein Purification - 1 Methods of Cell Lysis
Methods of Cell Lysis • Passive Methods: • Freeze-thaw • Chemical • Enzymatic • Osmotic Pressure • Physical Methods: • Sonication • Cell Bomb • Homogenizer Additional Considerations: • Volume • Cell Type • Toxicity • Cost • Further Purification Steps • Protein Stability • Protease activity/inhibitors • Protein location • pH • Salt • Reducing Agents
Chemical Lysis Adding a detergent to solubilize the cell membrane Detergent selection is critical!
Enzymatic Lysis Adding an enzyme to degrade the cell wall Protoplast
Enzymatic Lysis Lysozyme is most commonly used + H2O
Lysis by Osmotic Pressures Adjusting the concentration of electrolytes in the lysis buffer can promote lysis through osmotic pressures
PectateLyase Native organism Protein production organism PectateLyase is localized to the periplasm of E. coli
PectateLyase Strategy PectateLyase is localized to the periplasm of E. coli Purification Strategy Break the cell wall and expose the periplasmic space WITHOUT exposing cytosolic proteins
PectateLyase Strategy Purification Strategy Break the cell wall and expose the periplasmic space WITHOUT exposing cytosolic proteins Lyse 200 mMTris pH 8.3 500 mM Sucrose 5 mM EDTA 0.1 mg/mLLysozyme Wash 200 mMTris pH 8.3 Dialyze 20 mMTris pH 6.5
