ICBB Practical: Nuclear Preparation and Protein Analysis

Introduction to ICBB Practical: Nuclear Preparation & Isolation

Unit Number: U17658 (Introduction to Cell Biology and Biochemistry).
Primary Aims: * Analysis of blood smears to identify various blood cell types. * Isolation and preparation of nuclear and cytoplasmic proteins from erythrocytes.
Key Techniques Employed: * Microscopy: For blood smear analysis. * Centrifugation: Specifically differential centrifugation to separate cellular components based on size and density. * SDS PAGE (Polyacrylamide Gel Electrophoresis): To analyze the protein components extracted from the nucleus and cytoplasm. * Sample Preparation: Utilization of Triton X-100 to lyse cells and separate nuclei. * Staining: Use of Coomassie Blue dye to bind to and visualize proteins on the gel.

Haematopoiesis and Blood Cell Lineages

Haematopoiesis (or Hematopoiesis): The process by which all blood cells are formed from a common ancestor known as the Haemopoietic Stem Cell (HSC).
Stem Cell Characteristics: * HSC: Capable of self-renewal and differentiation into all blood lineages. * CFU-s: Colony-forming unit-spleen.
Lineage Progenitors: * Common Lymphoid Progenitor (CLP): Gives rise to lymphoid cells. * Pre-T cell: Develops into T-Lymphocytes. * Pre-B cell: Develops into B-Lymphocytes. * Common Myeloid Progenitor (CMP): Gives rise to myeloid cells. * MEP (Megakaryocyte-Erythroid Progenitor): * ER-CFC / BFU-E / CFU-E: Erythroid progenitors leading to Erythrocytes (Red Blood Cells). * Meg-CFC: Develops into Megakaryocytes, which produce Platelets. * GMP (Granulocyte-Monocyte Progenitor): * Eo-CFC: Leads to Eosinophils. * G-CFC: Leads to Neutrophils. * M-CFC: Leads to Monocytes/Macrophages. * GM-CFC: Combined progenitor for Granulocytes and Monocytes. * Mast-CFC: Leads to Mast cells.
Terminal Differentiation Products: * T-Lymphocytes and B-Lymphocytes. * Erythrocytes (RBCs). * Megakaryocytes / Platelets. * Eosinophils. * Neutrophils. * Monocytes / Macrophages. * Mast cells.

Avian Blood and Erythrocyte Development

Blood Source: Blood is analyzed from various species, though human and chicken blood are primary focus points. Other species mentioned include birds, amphibians, and camels.
Chicken $\beta$ -globin Expression: * Expression changes during development from embryo to adult. * Types of globin: $\beta^\rho$ , $\beta^A$ , $\beta^H$ , $\beta^\epsilon$ . * Timeline: Recorded at 5 days, 15 days, and 21 days (Hatching) after fertilization, and several weeks post-hatching. * Stages: Primary erythrocytes dominate early, replaced by mature erythrocytes later in development.
Erythrocyte Morphological Characteristics: * Primary Erythrocytes: Generally have a rounded appearance. * Mature Erythrocytes: Possess a distinctive oval shape. * Nucleation: Unlike human mature erythrocytes (which are anucleate), avian and amphibian erythrocytes maintain a nucleus.

Blood Smear Analysis of Chick Embryo Development

Incubation Age: 4 days 12 hours * 1–2: Mid-polychromatic primary erythrocytes. * 3–9: Late polychromatic primary erythrocytes. * 10–12: Late polychromatic erythrocytes demonstrating early cytosomal fracturing. * 13: Late polychromatic primary erythrocyte near metaphase. * 14: Late polychromatic primary erythrocyte in late telophase. * 15–16: Smudged primary erythrocytes. * 17: Later embryonic generation erythroblast(?). * 18–19: Embryonic thrombocytes.
Incubation Age: 9 days 15 hours * 1–3: Mature primary erythrocytes. * 4, 6–10: Late polychromatic erythrocytes. * 11–13: Late polychromatic erythrocytes with cytosomal fracturing. * 5, 14–18: Mature erythrocytes. * 19–20: Smudged erythrocytes. * 21: Embryonic thrombocytes (degenerated). * 22: Embryonic macrophage.
Incubation Age: 13 days 6 hours * 1: Mature primary erythrocytes. * 2–4: Mid-polychromatic erythrocytes with cytosomal fracturing. * 5–13: Late polychromatic erythrocytes. * 14–16: Mature erythrocytes. * 17: Microcyte. * 18: Erythroblast or thromboblast. * 19: Embryonic thrombocytes. * 20: Basophil mesomyelocyte.

Identification of Specific Blood Cells

Red Blood Cells (Erythrocytes): Cytoplasm is red to pink with a strongly staining purple/blue nucleus.
Neutrophils: Features dark purple nuclei and pale pink cytoplasm with reddish-lilac small granules.
Eosinophils: Features blue nuclei and pale pink cytoplasm with large red to orange-red granules.
Basophils: Features purple to dark blue nucleus with large granules that are dark purple to almost black.
Lymphocytes: Features dark purple to deep bluish-purple nuclei with sky-blue cytoplasm.
Platelets/Thrombocytes: Display violet to purple granules. Thrombocytes are the avian equivalent of megakaryocytes and may be seen in various stages of disintegration.

Principles of Centrifugation

Definition: Separation of biomolecules by spinning them through a solution using centrifugal force.
Sedimentation Coefficient ( $S$ ): Characterizes the behavior of a particle during sedimentation (measured in Svedbergs). * Formula: $S = \frac{\nu_t}{a}$ * $\nu_t$ = sedimentation speed ( $\text{m/s}$ ). * $a$ = applied acceleration ( $\text{m/s}^2$ ), which is the g-force related to rotations per minute (r.p.m.) for a specific rotor.
General Rule: Larger/denser particles sediment faster and have higher $S$ values.
Density vs. $S$ Value Comparison: * Soluble Proteins: Density ~ $1.35\,g\,cm^{-3}$ , $S$ value ~ $10^1$ . * RNA: Density ~ $2.0\,g\,cm^{-3}$ , $S$ value ~ $10^1$ . * DNA: Density ~ $1.7\,g\,cm^{-3}$ , $S$ value ~ $10^1$ to $10^2$ . * Ribosomes and Polysomes: $S$ value ~ $10^2$ . * Viruses: $S$ value ~ $10^2$ to $10^3$ . * Microsomes: $S$ value ~ $10^3$ . * Mitochondria: $S$ value ~ $10^4$ . * Chloroplasts: $S$ value ~ $10^4$ . * Nuclei: Density ~ $1.3\,g\,cm^{-3}$ , $S$ value ~ $10^6$ .
Nuclear Isolation Process: * Erythrocytes are treated with Triton X-100 detergent. * Differential centrifugation is applied. * The nuclear pellet is collected at the bottom of the tube. * The cytoplasm remains in the supernatant.

SDS PAGE: Principles and Theory

Function: Separation of proteins based primarily on size.
Role of SDS (Sodium Dodecyl Sulphate): * An anionic detergent consisting of a sulphate group ( $SO_3^-$ ) attached to a 12-carbon hydrocarbon chain. * The sulphate group interacts with water (hydrophilic), while the tail binds to hydrophobic regions of the protein.
SDS Interaction with Proteins: * Approximately 1 SDS molecule binds per every 2 amino acids. * SDS disrupts hydrophobic interactions within the protein core, between proteins in a complex, and between proteins and membrane lipids. * Proteins denature into an extended "random coil" or rod-like shape.
Charge Dynamics: * SDS provides a fixed charge:mass ratio of 1 negative charge per 2 amino acids. * This masks the intrinsic charge of the protein (determined by acidic groups like Asp/Glu and basic groups like Lys/Arg/His).
DNA Contrast: * DNA is naturally rod-like and has a fixed charge:mass ratio of 2 negative charges per base pair (bp). * DNA electrophoresis typically uses agarose gel, whereas proteins use polyacrylamide.
Gel Meshwork: Gels (agarose or acrylamide) act as a 3D meshwork of fibers with pores/channels. Larger molecules are retarded more by the sieving effect, resulting in separation by size.
Experimental Parameters: Run at $40\,mA$ current.

Protein Size Estimation

Molecular Weight Standards (Mw): Used to create a standard curve by plotting $\log(Mw)$ against relative migration.
Common Standards: * Myosin: $200,000\,Da$ * $\beta$ -Galactosidase: $116,250\,Da$ * Glycogen phosphorylase b: $97,400\,Da$ * Bovine serum albumin (BSA): $66,200\,Da$ * Ovalbumin: $45,000\,Da$ * Carbonic anhydrase: $31,000\,Da$ * Soybean trypsin inhibitor: $21,500\,Da$ * Lysozyme: $14,400\,Da$
Analysis: By comparing the relative migration of an unknown protein from the cytoplasm ( $S1$ ) or nuclei ( $N$ ) to the standards, the molecular weight of the unknown can be estimated.