Basic Electrophoresis Course

Lesson 1 • Historical Development of Electrophoresis

Traces key milestones from Tiselius moving-boundary work to modern gel systems. Provides context for understanding current technique diversity.

Lesson 2 • Electricity and Ionic Movement

Covers charge, voltage, current, and resistance as they apply to ions in solution. Connects Ohm's law to practical electrophoresis setup.

Lesson 3 • Buffers and pH Control

Teaches buffer selection, ionic strength, and pH stability during a run. Proper buffering is essential for reproducible separations.

Lesson 4 • Separation Driving Forces

Defines electrophoretic mobility and the forces opposing migration. Establishes the theoretical basis for band resolution.

Lesson 1 • Matrix Selection Criteria

Guides decisions between agarose and polyacrylamide based on molecule type and size. Reinforces the link between matrix choice and experimental goals.

Lesson 2 • Gel Preparation Techniques

Provides step-by-step protocols for casting both gel types safely and reproducibly. Emphasises degassing, levelling, and comb placement.

Lesson 3 • Gel Quality Assessment

Teaches visual and functional checks for gel integrity before loading samples. Poor gel quality is a leading cause of failed separations.

Lesson 4 • Agarose Gel Fundamentals

Describes agarose polymer structure, pore size, and concentration effects. Connects gel percentage to the size range of molecules resolved.

Lesson 1 • Running Conditions and Parameters

Defines voltage, run time, and buffer selection for nucleic acid gels. Optimising these parameters balances speed against resolution.

Lesson 2 • Troubleshooting Nucleic Acid Gels

Identifies causes of smearing, missing bands, and lane distortion. Systematic diagnosis prevents repeated experimental failures.

Lesson 3 • Sample Preparation for Nucleic Acids

Covers DNA and RNA quantification, purity assessment, and loading buffer composition. Proper sample prep prevents smearing and lane distortion.

Lesson 4 • Staining and Visualisation Methods

Compares ethidium bromide, SYBR Safe, and other intercalating dyes for nucleic acid detection. Addresses sensitivity, safety, and imaging requirements.

Lesson 1 • Molecular Weight Estimation

Teaches construction of standard curves from protein ladders to estimate unknown molecular weights. Accurate estimation requires proper ladder selection.

Lesson 2 • Gel System Design and Casting

Covers stacking and resolving gel composition, pH differences, and discontinuous buffer systems. Proper design sharpens band entry and resolution.

Lesson 3 • Protein Sample Preparation

Details lysis methods, protein quantification, and sample buffer preparation for SDS-PAGE. Consistent sample prep is critical for reproducible band patterns.

Lesson 4 • Running and Staining SDS-PAGE Gels

Provides protocols for electrophoresis conditions, Coomassie staining, and silver staining. Stain choice depends on sensitivity and downstream use.

Lesson 1 • Activity Staining After Native PAGE

Covers in-gel enzyme activity assays to identify functional proteins after native separation. Links band position to enzymatic function.

Lesson 2 • Blue Native PAGE Technique

Introduces Coomassie G-250 as a charge-shift agent in blue native PAGE for membrane proteins. This technique resolves protein complexes while maintaining native interactions.

Lesson 3 • Buffer and pH Selection for Native Gels

Guides pH and buffer choice to maintain protein charge and stability during native runs. Buffer conditions directly affect migration direction and band sharpness.

Lesson 4 • Principles of Native Electrophoresis

Contrasts native PAGE with SDS-PAGE, emphasising that separation depends on charge, size, and shape. Establishes when native conditions are required.

Lesson 1 • IPG Strip Rehydration and Loading

Details rehydration protocols, sample loading methods, and cup loading versus in-gel rehydration. Correct loading maximises protein entry and resolution.

Lesson 2 • Isoelectric Focusing Principles

Explains how proteins migrate to their isoelectric point in a pH gradient. IEF provides the first dimension of 2D electrophoresis.

Lesson 3 • IEF Running Conditions

Covers voltage ramping protocols, total volt-hours, and temperature control for IEF. Proper running conditions prevent protein precipitation at the pI.

Lesson 4 • 2D Gel Staining and Image Analysis

Covers fluorescent and colloidal staining for 2D gels and software-based spot detection. Quantitative analysis requires consistent staining and calibrated imaging.

Lesson 1 • Capillary Types and Coatings

Compares bare fused-silica, coated, and gel-filled capillaries for different analytes. Capillary selection determines EOF control and analyte interaction.

Lesson 2 • Detection Systems in CE

Covers UV absorbance, laser-induced fluorescence, and mass spectrometry detection for CE. Detector choice governs sensitivity and analyte identification capability.

Lesson 3 • CE Method Development and Optimisation

Guides buffer selection, voltage, and temperature optimisation for CE separations. Systematic method development reduces run time while maintaining resolution.

Lesson 4 • Capillary Electrophoresis Fundamentals

Introduces CE instrumentation, electroosmotic flow, and separation modes. CE offers higher resolution and automation compared to slab gel formats.

Lesson 1 • Documentation and Data Integrity

Establishes standards for image capture, file formats, and laboratory notebook records. Proper documentation supports reproducibility and regulatory compliance.

Lesson 2 • Statistical Analysis of Gel Data

Applies descriptive statistics, replicate analysis, and significance testing to electrophoresis results. Statistical rigour distinguishes real differences from experimental noise.

Lesson 3 • Quantitative Band and Spot Analysis

Teaches densitometry, lane normalisation, and background subtraction for gel images. Accurate quantification requires calibrated imaging and consistent protocols.

Lesson 4 • Common Artefacts and Their Causes

Catalogues gel artefacts including ghosting, satellite bands, and uneven migration. Recognising artefacts prevents misinterpretation of results.