SN6
SHORT NOTE
5M
★ High-yield
Describe the principle and clinical applications of electrophoresis.
Definition
Electrophoresis = the movement of charged particles in an electric field, used to separate proteins and nucleic acids according to their charge, size and shape.
Principle
- At a pH above the isoelectric point, a protein carries a net negative charge and migrates to the anode
- Serum electrophoresis is run at pH 8.6, where all serum proteins are negatively charged
- Rate of migration depends on net charge, molecular size and shape, the strength of the field, and the supporting medium
- Albumin moves fastest — it is the most negatively charged and relatively small
Supporting Media and Their Uses
| Medium | Separates by | Chief use |
|---|---|---|
| Cellulose acetate / agarose | Charge | Routine serum protein electrophoresis |
| SDS-page | Molecular size only — SDS masks the native charge | Determining molecular weight; Western blotting |
| Isoelectric focusing | Isoelectric point | Separating isoenzymes and haemoglobin variants; very high resolution |
| Two-dimensional | PI, then size | Proteomics |
| Agarose gel | Size | DNA and RNA |
| Capillary electrophoresis | Charge and size | Automated; increasingly replacing gels |
The Normal Serum Pattern
| Band | Percentage | Chief proteins |
|---|---|---|
| Albumin | 55–65% | Albumin |
| α1 | 2–4% | α1-antitrypsin |
| α2 | 6–12% | Haptoglobin, caeruloplasmin |
| β | 8–12% | Transferrin, C3 |
| γ | 12–22% | Immunoglobulins |
Diagnostic Patterns
| Condition | Characteristic finding |
|---|---|
| Multiple myeloma | Sharp M band in the γ region — monoclonal |
| Chronic infection or inflammation | Broad polyclonal ↑ γ |
| Cirrhosis | ↓ Albumin with β–γ bridging |
| Nephrotic syndrome | ↓↓ Albumin, ↑↑ α2, ↓ γ |
| α1-antitrypsin deficiency | Flat or absent α1 band |
| Agammaglobulinaemia | Absent γ band |
Related Techniques and Applied Aspects
- Immunofixation identifies the exact heavy and light chain type of an M band — essential in diagnosing myeloma
- Haemoglobin electrophoresis at alkaline pH separates HbA, HbF, HbS and HbC; the standard test for the haemoglobinopathies, which matter greatly in India
- Western blot uses SDS-page followed by antibody detection — the confirmatory test for HIV
- Isoenzyme separation — LDH and CK isoenzymes, used to localise tissue damage
- DNA electrophoresis underlies PCR analysis, Southern blotting and DNA fingerprinting
All questions in Chemistry of Lipids, Proteins & Amino Acids
- Q1Classify lipids with suitable examples. Describe their functions and the properties used to characterise them. ★
- Q2Classify fatty acids. Describe the essential fatty acids and the significance of trans fats. ★
- Q3Classify amino acids on the basis of side chain, nutritional requirement and metabolic fate. Describe their properties. ★
- Q4Describe the structural organisation of proteins from primary to quaternary level. Add a note on denaturation. ★
- Q5Classify the plasma proteins. Describe albumin and the acute phase response. Discuss the diagnostic value of serum electrophoresis. ★
- SN1Describe the structure, functions and clinical significance of cholesterol. ★
- SN2Describe the peptide bond with its characteristics and significance. ★
- SN3Describe the structure and types of haemoglobin. Add a note on its abnormal derivatives. ★
- SN4Describe the structure and biosynthesis of collagen. Add a note on its disorders. ★
- SN5Describe the structure, classes and functions of the immunoglobulins.
- SN6Describe the principle and clinical applications of electrophoresis. ★
- SN7Classify proteins on the basis of composition, shape and function. Add a note on precipitation reactions.