Chapter 5A: Molecular Tools for studying genes and gene activity

Gel Electrophoresis

Technique used to separate DNA, RNA, or proteins based on size and charge

DNA charge

DNA is negatively charged due to phosphate backbone

Direction of DNA migration

DNA moves toward the positive pole (anode) in a gel

Purpose of gel electrophoresis

To separate nucleic acids or proteins for analysis or purification

DNA staining

Fluorescent dye used to visualize DNA under UV light

Marker/ladder

DNA fragments of known size run alongside samples to estimate unknown sizes

Small DNA fragments

Move faster through the gel because they experience less friction

Large DNA fragments

Move slower through the gel because they experience more friction

PAGE

Polyacrylamide gel electrophoresis, used to separate proteins

SDS

Detergent that denatures proteins and gives them uniform negative charge

Effect of SDS on proteins

Proteins migrate according to size, not native charge

Reducing agents (e.g., B-mercaptoethanol)

Break disulfide bonds in proteins

Two-dimensional gel electrophoresis

Method to separate proteins by isoelectric point and size

Isoelectric focusing

Separation of proteins based on their isoelectric point (pH where net charge is 0)

Ampholytes

Molecules that create a pH gradient for isoelectric focusing

Second step of 2D electrophoresis

Proteins separated by SDS-PAGE after isoelectric focusing for size separation

Proteomics

Study of all proteins present in a cell or tissue at a given time