Preparation of Nuclear and Erythrocytes

  • Triton X-100

    • Used as a detergent for cell lysis in molecular biology.

  • Differential Centrifugation

    • A method to separate cellular components based on size and density.

  • Key Techniques:

    • Centrifugation:

    • Used to separate nuclei from cytoplasm.

    • Electrophoresis:

    • Utilized to analyze both nuclear and cytoplasmic components.

    • Includes methods such as SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

Centrifugation Overview

  • Definition:

    • The process of separating biomolecules by spinning through a solution at high speeds.

  • Sedimentation Coefficient (S):

    • Measured in Svedbergs (S), characterizes the behavior of particles during sedimentation.

    • Formula: S = rac{U t}{a}

    • Where:

      • S = Sedimentation coefficient

      • U = Sedimentation speed in m/s

      • a = Applied acceleration in m/s² (g force)

    • Can be related to revolutions per minute (r.p.m.) for each rotor.

  • Behavioral Dynamics:

    • Bigger particles sediment faster, leading to higher S values.

Sedimentation Details

  • Example of Sedimentation:

    • For a particle with a sedimentation coefficient of 26S (or 26 imes 10^{-13} ext{s} ), it will travel at a speed of 26 imes 10^{-6} ext{m/s} under an acceleration of 1 million gravities, approximately 10^7 ext{m/s}^2 .

  • Centrifugal Acceleration:

    • Given by the formula: a_c = r imes heta^2

    • Where r = radial distance from the rotation axis and heta = angular velocity in radians per second.

Density and Sedimentation Coefficient (S)

  • Density vs. S Value:

    • Density measurements for various biomolecules:

    • RNA: 2.1 g/cm³

    • DNA: 1.7 g/cm³

    • Ribosomes and polysomes

    • Soluble proteins: 1.5 g/cm³

    • Nuclei: 1.1 g/cm³

    • Most viruses: 1.3 g/cm³

    • Chloroplasts: 1.3 g/cm³

    • Mitochondria: 1.1 g/cm³

  • Sedimentation Coefficient Range:

    • Molecules range from 10 to 10^7 in their sedimentation rates.

Density Gradient Centrifugation

  • Technique Description:

    • EtBr-CsCl density gradient centrifugation processes DNA and RNA.

    • Steps include:

    • Shaking with n-butanol to allow for layer separation.

    • Removal of supercoiled DNA using a syringe.

    • Dialysis to remove EtBr and CsCl from the solution.

Sucrose Density Gradient Centrifugation

  • Concentration Levels:

    • 1 molar and 2 molar sucrose solutions utilized.

    • Example result from centrifugation:

    • Before: Samples mixed

    • After: Clear separation of lysosomes, mitochondria, peroxisomes, evident from specific densities.

Electrophoresis Overview

  • Definition: Gel electrophoresis is a technique predominantly used in molecular biology and biochemistry for:

    • Analyzing molecular weight and size of DNA, RNA, and proteins.

    • DNA sequencing and preparation/purification of DNA fragments for cloning.

Movement of Charged Particles in an Electric Field

  • Electrophoresis Dynamics:

    • Charged molecules migrate towards electrodes:

    • Positive molecules migrate to the negatively charged cathode.

    • Negative molecules migrate to the positively charged anode.

    • Migration is influenced by the force proportional to charge and resisted by frictional drag (dependent on size and shape).

Acid/Base Groups in Proteins

  • Amino Acids:

    • Basic building blocks of proteins with acidic (-COOH) and basic (-NH2) groups determining overall protein charge.

    • Common amino acids include:

    • Acidics: Aspartic acid (Asp), Glutamic acid (Glu)

    • Basics: Lysine (Lys), Arginine (Arg), Histidine (His)

Gel Electrophoresis Techniques

  • Principle of Separation:

    • Molecules are separated based on size in porous gels (agarose or acrylamide).

    • Larger molecules are obstructed by the sieving effect of the gel structure, resulting in size-based separation. Shape and charge can also play a role in this separation.

  • Discontinuous Buffer Systems:

    • Separates proteins by molecular weight using different pH buffers in gel and running buffer.

    • Low % acrylamide ‘stacking gel’ sharpens sample zones for improved resolution.

    • Challenges exist in ensuring proteins run independently of their native shape and charge.

Sodium Dodecyl Sulfate (SDS) Role in Electrophoresis

  • Chemical Composition and Function:

    • SDS is an anionic detergent composed of a sulfate group on a hydrocarbon chain (12 carbon atoms).

    • Causes denaturation of proteins, disrupting hydrophobic interactions both within the protein and with other proteins.

    • Proteins in SDS possess a uniform negative charge, swamping natural charges. The charge is dependent on protein length, resulting in extended random coil shapes during migration.

  • Outcome and Efficiency:

    • All proteins exhibit similar mobilities in free solutions due to denaturation by SDS. Choosing the correct gel matrix allows separation based on molecular size alone.

Acrylamide and Gel Composition

  • Regulating Pore Size:

    • The total acrylamide concentration and acryl/bisacrylamide ratio affect the pore sizes in the gel.

    • Varying these parameters optimizes separation capabilities of the gel during electrophoresis to target specific size ranges of proteins.

  • Polymerization Initiation:

    • Initiated by ammonium persulfate (S2O8^{2-}).

Stacking Gels and Electrode Buffer Systems

  • Structure of Gels:

    • Discontinuous gels employ electrode buffer systems to create a stack gel for improved separation:

    • Utilizes a low % acrylamide stacking gel to produce large pores with minimal sieving for the initial sample application.

    • Following this, a resolving gel with a different acrylamide percentage can effectively separate the proteins into narrow zones during electrophoresis.

Size Estimation of Proteins

  • Common Protein Standards:

    • Myosin: 200,000 Da

    • Beta-Galactosidase: 116,250 Da

    • Glycogen Phosphorylase b: 97,400 Da

    • Bovine Serum Albumin: 66,200 Da

    • Ovalbumin: 45,000 Da

    • Carbonic Anhydrase: 31,000 Da

    • Soybean Trypsin Inhibitor: 21,500 Da

    • Lysozyme: 14,400 Da

  • Formula for Molecular Weight Estimation:

    • Relative migration is correlated to size using the formula: 2 imes ext{log} Mw = M_r ext{(unknown)}

    • This allows unknown quantities to be correlated to standard proteins for estimation purposes.