Electrophoresis Course

Lesson 1 • Key Variables Affecting Separation

Identifies voltage, temperature, pH, and sample composition as critical parameters. Students learn to predict how changing each variable shifts migration patterns.

Lesson 2 • Historical Development and Applications

Traces electrophoresis from early serum protein studies to modern genomics. Contextualizes technique evolution and motivates subsequent technical chapters.

Lesson 3 • Principles of Charged Molecule Migration

Covers ionic charge, electric field interactions, and net charge concepts. Connects fundamental electrochemistry to why molecules move in a gel or solution matrix.

Lesson 1 • Specialty and Modified Gel Systems

Introduces gradient gels, denaturing gels, and low-melting-point agarose. Expands the student's toolkit for challenging separations encountered in later chapters.

Lesson 2 • Buffer Systems and Their Functions

Compares TAE, TBE, MOPS, Tris-glycine, and MES buffers by buffering range and ionic strength. Students match buffer choice to analyte type and downstream applications.

Lesson 3 • Polyacrylamide Gel Chemistry

Explains acrylamide and bis-acrylamide polymerization, crosslink density, and %T/%C notation. Prepares students to formulate gels for protein and small nucleic acid separations.

Lesson 1 • Molecular Weight Markers and Ladders

Explains ladder composition, fragment sizing, and proper loading amounts. Accurate size estimation depends on correct marker selection and lane placement.

Lesson 2 • Troubleshooting Common Gel Defects

Identifies causes of smearing, distorted lanes, faint bands, and background fluorescence. Systematic troubleshooting skills transfer to all gel-based techniques in later chapters.

Lesson 3 • Staining, Visualization, and Documentation

Compares ethidium bromide, SYBR Safe, and GelRed staining methods and UV/blue-light imaging. Students select safe, sensitive staining protocols and document results properly.

Lesson 4 • Sample Preparation for Nucleic Acids

Covers DNA and RNA quantification, loading buffer composition, and denaturation for RNA. Proper preparation prevents smearing and ensures reproducible migration.

Lesson 1 • Gel Formulation for SDS-PAGE

Covers stacking and resolving gel composition, acrylamide percentages, and gradient options. Students formulate gels matched to the molecular weight range of their target proteins.

Lesson 2 • Molecular Weight Estimation and Band Analysis

Uses protein ladders and linear regression on semi-log plots to estimate molecular weights. Students interpret band patterns to assess purity, degradation, and expression levels.

Lesson 3 • Running SDS-PAGE and Protein Staining

Guides students through assembly, electrophoresis conditions, and Coomassie or silver staining. Proper execution yields sharp, well-resolved bands suitable for downstream analysis.

Lesson 4 • Protein Sample Preparation

Details lysis methods, protein quantification assays, and sample buffer preparation. Consistent sample preparation is prerequisite to reproducible SDS-PAGE results.

Lesson 1 • Detection Systems and Signal Generation

Compares ECL chemiluminescence, fluorescent, and colorimetric detection methods. Students select detection systems based on sensitivity requirements and available instrumentation.

Lesson 2 • Blocking and Antibody Incubation

Covers blocking agents, primary antibody dilution optimization, and secondary antibody selection. Proper blocking and antibody conditions minimize background and maximize signal specificity.

Lesson 3 • Protein Transfer to Membranes

Compares wet, semi-dry, and dry transfer systems and PVDF vs. nitrocellulose membranes. Transfer efficiency directly determines detection sensitivity in subsequent steps.

Lesson 4 • Quantitative Western Blot Analysis

Introduces loading controls, normalization strategies, and densitometry software. Students produce statistically valid quantitative data from western blot images.

Lesson 1 • IPG Strip Rehydration and IEF Protocol

Details sample loading methods, rehydration conditions, and IEF voltage ramping programs. Correct protocol execution prevents horizontal streaking and ensures sharp focusing.

Lesson 2 • Principles of Native PAGE

Explains separation by charge, size, and shape under non-denaturing conditions. Students understand when native PAGE is preferred over SDS-PAGE for functional studies.

Lesson 3 • Isoelectric Focusing Fundamentals

Explains pH gradient formation, protein focusing at pI, and carrier ampholyte vs. IPG strip systems. IEF resolves proteins differing by as little as 0.01 pH units.

Lesson 4 • Two-Dimensional Gel Electrophoresis

Combines IEF and SDS-PAGE to resolve thousands of proteins by pI and molecular weight. Students execute 2-DE workflows and interpret complex spot patterns.

Lesson 1 • Capillary Gel Electrophoresis for Nucleic Acids

Applies polymer-filled capillaries to DNA fragment sizing and RNA quality assessment. CGE replaces slab gels with automated, quantitative, high-throughput analysis.

Lesson 2 • Capillary Zone Electrophoresis

Covers CZE separation principles, buffer selection, and injection modes for small molecules and proteins. Students develop and validate CZE methods for defined analyte classes.

Lesson 3 • Capillary Electrophoresis Instrumentation

Describes capillary dimensions, high-voltage power supplies, detectors, and autosampler components. Instrument familiarity is prerequisite to method development in subsequent sections.

Lesson 4 • Capillary Isoelectric Focusing

Adapts IEF principles to capillary format for protein pI determination and isoform profiling. Students compare cIEF to gel-based IEF in resolution and throughput.

Lesson 1 • Method Development and Validation

Applies design-of-experiments principles to optimize gel percentage, buffer, and run conditions. Validation parameters include specificity, linearity, range, and reproducibility.

Lesson 2 • Quantitative Densitometry and Image Analysis

Covers image acquisition settings, background subtraction, and lane profile analysis for accurate quantification. Students use software tools to extract reliable numerical data from gel images.

Lesson 3 • Pulsed-Field Gel Electrophoresis

Explains alternating electric field principles enabling separation of megabase DNA. Students apply PFGE to microbial typing and large-genome analysis.

Lesson 4 • Integration with Downstream Analytical Methods

Connects electrophoresis outputs to mass spectrometry, sequencing, and functional assays. Students design multi-step workflows where electrophoresis serves as a preparative or analytical step.