Unit 3: Electrophoresis
Analytical Biochemistry notes · PTU syllabus (BMLS302-18)
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Unit summary
Electrophoresis separates charged molecules in an electric field. This unit covers its principle, instrumentation, types — paper and gel electrophoresis — and applications.
After this unit you can
- Explain the principle of electrophoresis
- Describe the instrumentation and factors affecting mobility
- Describe paper, agarose, PAGE and SDS-PAGE electrophoresis
- Interpret serum protein and haemoglobin electrophoresis
PTU syllabus topics
- Introduction
- principle
- instrumentation
- types (paper and gel electrophoresis) and applications
- 1Apply serum to the gel or strip
- 2Apply voltage
Proteins move by charge
- 3Stain the bands
- 4Read the pattern
Albumin moves farthest
- 5Bands
Albumin, α1, α2, β, γ globulins
Topic 1
Principle of electrophoresis
- Charged particles in a buffer move towards the electrode of opposite charge when a voltage is applied. Mobility depends on net charge, size and shape, the buffer pH and ionic strength, voltage, and the support medium.
- At pH 8.6, serum proteins are negatively charged and move towards the anode (+).
Topic 2
Instrumentation
- 1
Power pack (constant voltage or current)
- 2
Buffer tanks with electrodes (barbital buffer pH 8.6 for serum proteins)
- 3
Support medium bridging the tanks — paper, cellulose acetate, agarose, polyacrylamide
- 4
Sample applied at the origin; run for set time
- 5
Fix and stain (Ponceau S, amido black, Coomassie blue)
- 6
Densitometer scans bands for quantitation
Topic 3
Types of electrophoresis
Paper
Simple, slow, more diffusion
Historical serum proteins
Cellulose acetate
Fast, clear bands
Serum proteins, haemoglobin
Agarose gel
Large pores, non-toxic
Serum proteins, DNA, lipoproteins, immunoelectrophoresis
Polyacrylamide gel (PAGE)
Small adjustable pores; high resolution
Proteins, isoenzymes
SDS-PAGE
SDS gives uniform negative charge; separates by molecular weight
Protein size, western blotting
Isoelectric focusing
pH gradient; proteins stop at their pI
High-resolution protein and Hb variants
Capillary electrophoresis
Narrow capillaries, high voltage, automated
Serum proteins, HbA1c
Topic 4
Applications
Albumin (largest band, nearest anode)
About 55–65%
Low in nephrotic syndrome, cirrhosis
α1-globulin
α1-antitrypsin
Low in α1-antitrypsin deficiency
α2-globulin
Haptoglobin, α2-macroglobulin
Raised in nephrotic syndrome and inflammation
β-globulin
Transferrin, complement
Raised in iron deficiency
γ-globulin
Immunoglobulins
Sharp M-band in multiple myeloma; broad rise in chronic infection and cirrhosis
- Haemoglobin electrophoresis
- HbS (sickle cell), HbA2 raised in β-thalassaemia trait
- Isoenzymes
- LDH and CK isoenzymes
- Lipoprotein electrophoresis
- Hyperlipoproteinaemia types
- Nucleic acids
- DNA fragments in agarose — PCR products, DNA fingerprinting
- Immunofixation
- Typing monoclonal proteins
Key terms
- Electrophoresis
- Movement of charged particles in an electric field
- Anode
- Positive electrode
- SDS-PAGE
- Protein separation by size in polyacrylamide gel with SDS
- Densitometer
- Instrument scanning stained bands
- M-band
- Narrow monoclonal immunoglobulin band
Quick revision
- Principle; factors affecting mobility; pH 8.6 serum proteins to anode.
- Power pack, buffer, support, staining, densitometry.
- Paper, cellulose acetate, agarose, PAGE, SDS-PAGE, IEF, capillary.
- Five serum protein fractions; myeloma M-band; nephrotic pattern.
- Hb electrophoresis; isoenzymes; DNA.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.State the principle of electrophoresis.
- Q2.Why do serum proteins move to the anode at pH 8.6?
- Q3.What is the role of SDS in SDS-PAGE?
- Q4.Name the five serum protein fractions.
- Q5.What is an M-band?
- Q6.Give one use of agarose gel electrophoresis.
Long-answer questions
- Q1.Explain the principle and instrumentation of electrophoresis.
- Q2.Describe the types of electrophoresis.
- Q3.Describe serum protein electrophoresis and its clinical interpretation.
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