SDS-PAGE: Protein Separation Technique

SDS-PAGE is a technique used to separate proteins based on their molecular weights.
The process involves several steps: sample preparation, loading, electrophoresis, separation by size, visualization, and interpretation.

Proteins are denatured and coated with SDS, an anionic detergent that binds to proteins and unfolds them into linear chains.
SDS masks the intrinsic charge of proteins and provides a uniform negative charge based on the mass of the polypeptide chain.

The gel is composed of a cross-linked matrix of polyacrylamide, which forms a porous network that allows proteins to migrate through it based on their size.
The gel has two regions: stacking gel and separating gel, each serving a specific purpose in facilitating protein separation.

Purpose: concentrates and focuses protein samples into a narrow zone before they enter the separating gel.
Composition: lower percentage of acrylamide (around 4-5%) creates a more porous matrix.
Buffer System: high-resistance environment slows down protein movement, leading to protein stacking at the interface between the stacking gel and the separating gel.

Purpose: separates proteins based on their molecular weight.
Composition: higher percentage of acrylamide (8-15%) allows for improved resolution of proteins.
Gradient Gels: acrylamide concentration gradually increases from top to bottom, allowing for improved resolution of proteins across a wider range of molecular weights.

An electric field is applied to the gel, causing proteins to migrate through the gel matrix based on their size.
Smaller proteins move more quickly and travel farther through the gel, while larger proteins move more slowly and remain closer to the point of origin.

Osmosis is the movement of solvent molecules across a semi-permeable membrane from an area of lower solute concentration to an area of higher solute concentration.
This process occurs spontaneously and does not require energy.

Hypertonic Solution: has a higher concentration of solutes compared to another solution, causing water to move out of cells and leading to cellular dehydration.
Hypotonic Solution: has a lower concentration of solutes compared to another solution, causing water to move into cells and leading to swelling or lysis.
Isotonic Solution: has the same concentration of solutes as another solution, resulting in no net movement of water and maintenance of cell shape and size.

Hypertonic Solution: isopropanol solutions can be hypertonic compared to the cytoplasm of RBCs, causing cellular dehydration.
Hypotonic Solution: pure water is a hypotonic solution, causing water to rush into cells and leading to swelling or lysis.
Isotonic Solution: normal saline (0.9% NaCl) is an isotonic solution, maintaining the normal shape and size of cells.

Four important steps are required for successful nucleic acid purification: effective disruption of cells or tissue, denaturation of nucleoprotein complexes, inactivation of nucleases, and extraction of nucleic acids.

Guanidinium Thiocyanate-Phenol-Chloroform Extraction is a conventional method of nucleic acid extraction.
This method uses phenol: chloroform: isoamyl alcohol (25:24:1) to remove proteins, lipids, carbohydrates, and cell debris.
Guanidinium thiocyanate is a chaotropic agent used in protein degradation.
The hydrophobic layer of the emulsion settles on the bottom and the hydrophilic layer on top by centrifugation.
The upper phase containing DNA is collected and DNA can be precipitated from the supernatant by adding ethanol or isopropanol in 2:1 or 1:1 ratios and high concentration of salt.
DNA precipitate is collected by centrifugation, and excess salt is rinsed with 70% ethanol and centrifuged to discard the ethanol supernatant.

In acidic conditions, total RNA remains in the upper aqueous phase of the whole mixture.
DNA and proteins remain in the interphase or lower organic phase.
Recovery of total RNA is done by precipitation with isopropanol.

The principle of the method is based on selective alkaline denaturation of high molecular weight chromosomal DNA.
Plasmid DNA can be recovered from the supernatant after the denatured material has been removed by centrifugation.

CTAB (Cetyltrimethylammonium bromide) is a nonionic detergent used for purification of nucleic acid from organisms that produce large quantities of polysaccharides.
CTAB can precipitate nucleic acids and acidic polysaccharides from low ionic strength solutions.
Proteins and neutral polysaccharides remain in solution under these conditions.

Transmittance, absorbance, and extinction coefficient are key concepts in spectrophotometry.
The extinction coefficient is an intrinsic property of the isolates that determines how strongly a species absorbs or reflects radiation or light at a particular wavelength.
The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution.

Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool.
The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v).
Agarose gels are normally in the range of 0.2% to 3%.
The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes like bromophenol blue and xylene cyanol.

Affinity purification is an important step in protein purification.
SDS-PAGE gel electrophoresis is used for the separation of proteins based on charge/mass (e/m) ratio.