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Agarose gel electrophoresis and interpretation
Practical aspect of agarose gel electrophoresis and interpretation of electrophoresis patterns.
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Agarose gel electrophoresis and interpretation
- 1. ELECTROPHORESIS PROF. VIYATPRAJNA ACHARYA MD,PhD Dept of Biochemistry, KIMS & PBH, Bhubaneswar
- 2. ELECTRO + PHORESIS •Movement of charged particle through an electrolyte when subjected to an electrical field • Tiselius (NP-1948)- discovered • Mixture of charged particles can be separated • Positively charged particles will move to cathode-cations • Negatively charged particles will move to anode- anions
- 3. PROTEINS, LIPOPROTEINS ANDHEMOGLOBINVARIANTS CAN BE SEPARATED 1. Agarose gel electrophoresis 2. Cellulose acetate electrophoresis 3. Paper electrophoresis 4. Polyacrylamide gel electrophoresis 5. Disc electrophoresis 6. Isoelectric focusing 7. Two-dimensional electrophoresis 8. Capillary electrophoresis 9. Immunoelectrophoresis Types: Disc electrophoresis
- 4. AGAROSE GEL ELECTROPHORESIS (FORPLASMA PROTEIN SEPARATION) Requisites: 1. Electrophoresis chamber 2. Power pack- options of constant voltage and current should be there 3. Barbitone buffer (pH- 8.6)/TAE Buffer (Tris-Acetate-EDTA)/ TBE Buffer (Tris-Borate-EDTA), NaOH-Boric acid 4. Agarose powder- 100 ml of agarose powder in 10 ml of buffer 5. Tagging dye- 1 ml buffer saturated with Bromophenol blue 6. Fixative- 70% ethanol 7. Staining solution- 0.5 gm amido black 10B in 100 ml 5% acetic acid 8. Glass slide 9. Whatman filter paper 3
- 5. PROCEDURE: • Take theagarose powder in buffer and boil it while stirring intermittently till it is fully dissolved. • Cool it to 60-700 c and pipette 1.2-1.4 ml of the gel unto the slide from one side and allow it to spread evenly. • One drop of serum is mixed with the tagging dye. • From one side of the slide the serum is applied with the help of an applicator or a strip of filter paper. • The buffer tanks are filled with the buffer. • Whatman 3 filter paper strips are cut into pieces same as the width of glass slide. Contact is made to the buffer and gel plate by means of the filter paper strips. • Direct current is passed for one hour at 150-180V and 5mA. • Slides were taken out and dipped in fixative solution for 10-15 minutes.Take out the slides and dry them at 700 c till exactly dry. • Then dip the slides in staining solution for 10 minutes and washed with 5-10% acetic acid and air dry the slides. Observe the different bands.
- 7. FACTORS AFFECTING ELECTROPHORESIS •size of charged particles, • charge of ions, • pH of the medium, • strength of the electric field • temperature of operation. • Properties of the supporting medium
- 8. INTERPRETATION • In normalelectrophoretogram normally 5 bands are seen. From cathode to anode they are- • - γ globulin (12%), • - β globulin (15.5%), • -2 α globulin (13.5%), • -1 α globulin (3%) • albumin (56%) • Serum electrophoresis helps in diagnosing different disease conditions. For example, in nephrosis albumin level decreases and -2 α globulin level increases. In Multiple myeloma an extra band known as M-band appears between β and γ bands.
- 10. CIRRHOSIS • Alb decreased •Beta- often increased • Gamma- markedly increased • Beta-gamma bridging
- 11. NEPHROTIC SYNDROME • Albuminmarkedly decreases • Prominent Alpha 2 band • - β globulin rise • -globulin decrease γ (contrast this with cirrhosis or chronic inflammation where increases). γ
- 12. ACUTE INFLAMMATION • ProminentAlpha 1 &2 • Gamma may rise • Albumin may decrease
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- 16. FAR ADVANCED CIRRHOSIS;ẞ-Ý FISSION, ALBUMIN ↓
- 17. 1.Normal pattern 2.Multiple myeloma-M band 3.Chr infection- broad based gamma; increase in alpha 1 & 2 4.Nephrotic syndrome- Low albumin; prominent alpha 2 5.Chronic cirrhosis- decreased albumin 6.Normal with fibrinogen band
- 18. • 2,4, 8& 10- Normal pattern • 1-Nephrotic syndrome • 3- Cirrhosis; bridging • 5- Chronic infection- broad based gamma • 6,7- Multiple myeloma • 9- Acute inflammation- less albumin, increased alpha-2
- 19. LIPOPROTEIN ELECTROPHORESIS • OriginChylomicrons → → ( β LDL) pre- → ( β VLDL) → ( α HDL) Anode → Condition Electrophoresis Pattern Type I Hyperlipoproteinemia Prominent chylomicron band Type IIa (Familial Hypercholesterolemia) Broad, intense band (LDL) β Type IIb Broad and pre- bands (LDL +VLDL) β β Type III (Dysbetalipoproteinemia) Broad β band ( - β VLDL) Type IV (Familial Hypertriglyceridemia) Prominent pre-β band (VLDL) TypeV Chylomicrons + increased pre- ( β VLDL)
- 20. HB ELECTROPHORESIS HemoglobinType Migration Speed(toward Anode) Notes HbA Fast Normal adult hemoglobin HbF Slower than HbA Fetal hemoglobin HbS Slower than HbA Seen in sickle cell disease HbC Even slower than HbS Seen in HbC disease
- 21. FOR MORE PPTON MEDICAL BIOCHEMISTRY Please visit www.drvpacharya.com