Protein Study and Purification

Introduction to Protein Extraction

• Source of Proteins: Commonly obtained from cells.
• Cell Fractionation: Process to extract proteins from cells.

Steps in Cell Fractionation

1. Forming a Homogenate:
   • Cells are placed in a test tube and exposed to grinding or mixing.
   • This ruptures cell membranes, mixing components like nuclei, organelles, ribosomes, etc.
2. Differential Centrifugation:
   • Separates components based on density using centripetal acceleration.
   • Denser components settle lower in test tube (pellet), less dense remain higher (supernatant).

Centrifugation Process

• First Centrifugation:
  • Acceleration: 1,000 G's.
  • Result: Nuclei in pellet, rest in supernatant.
• Second Centrifugation:
  • Acceleration: 10,000 G's.
  • Result: Mitochondria in pellet.
• Third Centrifugation:
  • Acceleration: 100,000 G's.
  • Result: Microsomes in pellet, proteins in supernatant.

Protein Assay

• Conducted at every step to identify protein activity.
• Identifies the fraction with the highest protein activity for further use.

Protein Purification

• Properties for Separation:
  • Size
  • Solubility
  • Charge
  • Binding ability to biological molecules

Types of Purification Processes

1. Dialysis: Separates proteins from small molecules using a semipermeable membrane.
2. Salting Out: Separates proteins based on their precipitation at specific salt concentrations.
3. Gel Filtration Chromatography: Uses size to separate proteins, larger proteins move faster.
4. Ion Exchange Chromatography: Separates proteins based on charge.
5. Affinity Chromatography: Uses binding affinity to separate proteins.
6. Gel Electrophoresis: Separates proteins based on size using electric field.
7. Isoelectric Focusing: Separates proteins based on isoelectric point (pH where net charge is zero).
8. Two-Dimensional Electrophoresis: Combines isoelectric focusing and gel electrophoresis.

Importance of Enzyme Activity and Concentration

• Determines specific activity.
• Higher specific activity indicates greater purity of sample.
• Successful purification processes increase specific activity value.