ICBB Practical: Nuclear Preparation and Protein Analysis

Flashcards covering blood cell types, hematopoiesis, protein chemistry, and laboratory techniques such as centrifugation and SDS PAGE based on the ICBB practical notes.

Practical Aims

Blood Smear Analysis and nuclear preparation & isolation of cytoplasmic & nuclear proteins.

HSC

Haemopoietic stem cells.

CLP

Common lymphoid progenitors.

CMP

Common myeloid progenitor cells.

Primary erythrocytes

Cells with a rounded appearance and a strongly staining purple/blue nucleus, seen in early chick embryo development stages (e.g., 4d 12h).

Mature erythrocytes

Cells with a distinctive oval shape and red to pink cytoplasm with a strongly staining purple/blue nucleus.

Neutrophils

Cells characterized by dark purple nuclei, pale pink cytoplasm, and reddish-lilac small granules.

Eosinophils

Cells characterized by blue nuclei, pale pink cytoplasm, and red to orange-red large granules.

Basophils

Cells characterized by purple to dark blue nucleus and dark purple, almost black, large granules.

Lymphocytes

Cells characterized by dark purple to deep bluish purple nuclei and sky blue cytoplasm.

Thrombocytes

The avian equivalent of megakaryocytes, observed in various stages of disintegration.

Chicken $\beta$-globin expression

The expression of globin types including $\beta\rho$, $\beta\text{A}$, $\beta\text{H}$, and $\beta\text{ε}$ during development from 5 days fertilization to weeks after hatching.

Centrifugation

A technique used to separate nuclei from cytoplasm or biomolecules by spinning through solution based on size and density.

Sedimentation coefficient (S)

Characterises the behaviour of a particle during sedimentation and is defined by the formula $S = \frac{\nu_t}{a}$, where $\nu_t$ is sedimentation speed in $ms^{-1}$ and $a$ is applied acceleration in $ms^{-2}$.

Triton X-100

A reagent used in the preparation of nuclear and cytoplasmic extracts from erythrocytes.

Coomassie

A blue dye which binds to protein used to stain gels in SDS PAGE.

DNA structure charge:mass ratio

A fixed ratio of $2-ve$ per base pair, with a distance of $0.34\text{ nanometers}$ between each base pair.

Acidic amino acids

Amino acids including Aspartate (Asp) and Glutamate (Glu), containing $-COOH$ groups.

Basic amino acids

Amino acids including Lysine (Lys), Arginine (Arg), and Histidine (His), containing $-NH_2$ groups.

SDS (sodium dodecyl sulphate)

An anionic detergent with a hydrocarbon chain of $12\text{Cs}$ and a sulphate group ($SO_3^-$) that denatures proteins and provides a fixed charge:mass ratio of $1-ve/2\text{ amino acids}$.

SDS PAGE

Polyacrylamide gel electrophoresis using sodium dodecyl sulphate to separate denatured proteins by size.

Gel Electrophoresis principle

Separates molecules principally by size using a 3D meshwork of agarose or acrylamide fibres with 'pores' or channels which retard larger molecules.

Bovine serum albumin

A protein size estimation standard with a molecular weight of $66,200$.

Myosin

A protein size estimation standard with a molecular weight of $200,000$.

Lysozyme

A protein size estimation standard with a molecular weight of $14,400$.