Overview
Electrophoresis is the movement of charged particles under a spatially uniform electric field in a fluid. First observed by Ferdinand Frederic Reuss in 1807 with clay particles migrating in water, it is now one of the most widely used analytical and separation tools in biochemistry, capable of separating DNA, RNA, and protein molecules by size, charge, or binding affinity.
As an analytical tool, electrophoresis is simple, rapid, and highly sensitive. It forms the basis of numerous laboratory techniques β from routine DNA gel electrophoresis after PCR amplification to protein separation using SDS-PAGE β and is indispensable in molecular biology, clinical diagnostics, and forensic science.
Learning Objectives
After this lesson you will be able toβ¦- Define electrophoresis and describe the physical principle behind particle migration in an electric field.
- Describe the principle and important types of electrophoretic methods.
- Explain the principle and components of agarose gel electrophoresis.
- List the equipment, reagents, and step-by-step procedure for running a standard agarose gel.
- Explain the various clinical and research uses of electrophoresis.
Clinical Story
Why This MattersA newborn is suspected of having a hemoglobinopathy after a routine screening shows abnormal red cell morphology. The physician orders hemoglobin electrophoresis to separate and identify abnormal hemoglobin variants such as HbS or HbC. The lab technologist must know how to run the gel correctly, apply the right voltage, and interpret band migration patterns to support an accurate diagnosis β a skill built on the fundamentals of electrophoresis covered in this lesson.
Core Concepts
Charged particles in a fluid are surrounded by a diffuse layer of oppositely charged ions (the double layer theory). When an external electric field is applied, it exerts a force on these ions, part of which is transferred to the particle surface as the "electrophoretic retardation force." The particle's velocity is proportional to the applied field, giving its electrophoretic mobility (ΞΌe = Ξ½/E). The Smoluchowski theory (1903) is the most widely used model, valid when the particle radius is much greater than the Debye length (a thin double layer, aΞΊ >> 1). The Huckel equation applies instead when the double layer is thick (aΞΊ < 1).
Major types include: Affinity electrophoresis (mobility shift, charge shift, based on biospecific interactions); Capillary electrophoresis (CE) β separates ionic species by charge and hydrodynamic radius inside a small capillary; Immunoelectrophoresis β combines electrophoresis with antibody reactions (rocket, fused rocket, affinity types); Pulsed field gel electrophoresis (PFGE) β separates very large DNA molecules using a periodically changing electric field direction; SDS-PAGE β separates proteins by molecular size after denaturation with SDS detergent; Native gels β proteins run without denaturation, so native charge determines migration; Electrofocusing gels β separate proteins along a pH gradient until they reach their isoelectric point; DNA agarose gels β separate large DNA fragments by size using the constant charge-to-mass ratio of the phosphate backbone.
Fragments of linear DNA migrate through agarose gel with a mobility inversely proportional to the log10 of their molecular weight. Plotting migration distance against log10 of molecular weight (or base pairs) gives a roughly straight line β the basis of estimating unknown fragment sizes against a DNA ladder. Bromophenol blue and xylene cyanol tracking dyes migrate at roughly the same rate as double-stranded DNA fragments of 300 bp and 4000 bp respectively, letting technicians visually monitor how far electrophoresis has proceeded.
Laboratory Principle
DNA and RNA molecules carry a uniformly negative charge from their phosphate backbone, so under an applied electric field they migrate toward the positive electrode (anode) at a rate inversely related to the log of their molecular weight. Smaller fragments move faster through the pores of the agarose or polyacrylamide matrix; larger fragments are retarded. Ethidium bromide intercalates between DNA base pairs and fluoresces under UV light, allowing visualization of separated fragments on a transilluminator.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Agarose powder | 0.8% (0.14 g in 20 mL buffer) | Forms the gel matrix for DNA separation | Room temperature, dry |
| TAE buffer (1X) | Tris-acetate-EDTA | Electrophoresis running buffer | Room temperature |
| Ethidium bromide (EtBr) | 1%, final gel conc. 0.5 Β΅g/mL | Fluorescent nucleic acid stain | Dark, hazardous β handle with gloves |
| 6X loading dye | Contains Bromophenol blue & glycerol | Adds density to load sample and tracks migration | 4Β°C |
Step-by-Step Procedure
For a 0.8% gel, dissolve 0.14 g of agarose in 20 mL of 1X TAE buffer. Boil until a clear solution is obtained.
Leave the solution at room temperature until it reaches 40β45Β°C, then add 2 Β΅L of 1% ethidium bromide.
Seal the casting tray, place the comb, pour the gel, and leave at room temperature for 45β50 minutes to solidify.
Fill the buffer tank with 1X TAE so the gel is submerged. Load 5 Β΅L of sample mixed with 1 Β΅L of 6X loading dye into each well.
Switch on the power supply at 5V/cm. When the dye front reaches the bottom of the gel, switch off the power, place the gel on the transilluminator, and observe under UV light with protective eyewear.
Flow Diagram
Quality Control
Always run a molecular weight marker (DNA ladder) alongside samples so band sizes can be estimated. Include a known positive control to confirm the gel and buffer system are working, and monitor bubble formation at the electrodes to confirm current is flowing correctly.
Laboratories performing diagnostic electrophoresis (e.g., hemoglobin or protein electrophoresis) should participate in external proficiency testing schemes to verify accuracy of band pattern interpretation against peer laboratories.
Reference Values
Typical Run Parametersβ οΈ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Single diffuse high-MW band | DNA fragments above 30β50 kb threshold running together | Use pulsed field gel electrophoresis (PFGE) instead |
| Sharp, distinct bands at expected size | Successful amplification/digestion, expected fragment pattern | Proceed with downstream application (sequencing, cloning) |
| Smeared bands or no bands | Degraded DNA, poor loading, or run/voltage error | Repeat extraction, check sample integrity and voltage settings |
Common Errors & How to Avoid Them
Cause: Comb removed too soon or too forcefully before the gel fully solidifies.
Prevention: Allow the gel to solidify fully (45β50 minutes) and remove the comb slowly and vertically.
Cause: Voltage too high, run time too long, or gel percentage inappropriate for fragment size.
Prevention: Use 5V/cm as a standard rate, monitor tracking dye migration, and adjust agarose percentage to the fragment size range expected.
Cause: Handling EtBr without gloves, or viewing the transilluminator without eye protection.
Prevention: Always wear gloves when handling EtBr (known mutagen) and protective eyewear when using the UV transilluminator.
Laboratory Tips from the Bench
Air bubbles trapped near the wells while loading can cause sample to leak out β tap the tray gently to dislodge bubbles before loading.
Always confirm current is flowing correctly by watching for bubbles forming at the electrodes before walking away from the run.
"DNA runs to Red" β the red electrode is the positive anode, and negatively charged DNA always migrates toward it.
Important Notes
EtBr is a known mutagen and must be handled as a hazardous chemical at all times β always wear gloves and dispose of EtBr-contaminated waste according to institutional biohazard protocols.
The Smoluchowski theory applies to a "thin double layer" (particle radius >> Debye length) and is valid for most aqueous systems. The Huckel equation applies to a "thick double layer," useful for nanoparticles and non-polar fluids.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeAnika presents with pallor, poor growth, and mild jaundice. Her parents are both carriers of an unspecified hemoglobin trait. The pediatrician orders hemoglobin electrophoresis to identify the type and proportion of hemoglobin variants present.
The electrophoretic pattern shows a predominant abnormal HbS band with reduced HbA, consistent with sickle cell disease rather than trait. Electrophoresis separates hemoglobin variants based on their differing net charge, which alters migration distance in the electric field.
- βElectrophoresis separates hemoglobin variants by differences in net surface charge.
- βA predominant HbS band with low/absent HbA distinguishes disease from trait.
- βCorrect interpretation requires understanding of band migration principles taught in this lesson.
Frequently Asked Questions
DNA's phosphate backbone carries a uniform negative charge, so in an electric field it is attracted toward the positive electrode (anode), which is usually colored red on electrophoresis equipment.
Agarose gels have larger pores and are used for separating larger DNA/RNA fragments, while polyacrylamide gels have finer pores suited to resolving smaller fragments (sequencing gels) or proteins (SDS-PAGE), down to a resolution of a single base pair difference.
SDS denatures proteins and coats them uniformly with negative charge proportional to their length, so that separation depends only on molecular size as the protein passes through the polyacrylamide matrix, not on native charge or shape.
Quick Revision
10-Minute ReviewKey Takeaways
- Electrophoresis plays a vital role in the separation of nucleic acids and proteins in genomics and proteomics.
- The technique is simple but has broad applications in advanced research and clinical diagnostics.
- Electrophoretic devices are economical and versatile enough to analyze the complexity of biomolecules.
- Different types of electrophoresis (SDS-PAGE, native gels, electrofocusing, PFGE, capillary electrophoresis) suit different separation needs.
- Correct handling of ethidium bromide and UV light protects both sample integrity and technician safety.
Competency Checklist
Track Your MasteryReferences
- NIOS Biochemistry Module β Lesson 21: Electrophoresis.
- Smoluchowski, M. (1903). Theory of electrokinetic phenomena.
- Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual.