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Isoelectric Focusing
This document provides an overview of isoelectric focusing (IEF), a technique used to separate amphoteric molecules like proteins based on their isoelectric point (pI). IEF involves subjecting proteins to an electric field within a pH gradient formed in a polyacrylamide gel. Proteins will migrate within the gel until they reach the point where they are electrically neutral (pI) and stop migrating. Different proteins have different pIs and so become separated. Key steps include establishing a stable pH gradient using ampholytes, loading protein samples mixed with loading buffer, running the gel at increasing voltages, and then fixing and staining to visualize separated protein bands. IEF can also be used in the first dimension
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Isoelectric Focusing
- 1. ISOELECTRIC FOCUSINGM. KALEEM IQBAL 0309-MPHIL-BIO-T-12 INSTITUTEOF INDUSTRIAL BIOTECHNOLOGY GCU, LAHORE.
- 2. Isoelectric Focusing Processby which amphoteric molecules can be separated on the basis of their isoelectric points. Basic principle involved is electrophoresis. Proteins are subjected to electric field in a pH gradient. Requires a solid surface normally Polyacrilamide.
- 3. Iso-electric Point (pI) ThepH at which net charge on protein becomes zero. Below pI = Positive Charge. Above pI = Negative Charge. Proteins move toward the electrode with the opposite charge. During motion, proteins will either pick or loose protons. Different from conventional electrophoresis migrate to steady state.
- 4. Ampholytes Establishment ofstable pH gradient is important. Achieved by means of commercially available synthetic carrier amphoteric electrolytes. 600 – 900 Da. Closely spaced pI and high conductivity. The curve is determined by pH interval covered by the ampholyte and the distance between electrodes.
- 5. Working Procedure. FollowingChemicals are required: Acrylamide solution. Water. Ampholyte solution pH 3.5 – 10. Ampholyte solution pH 4 – 6. Urea. Spin gently to mix urea. Add 10% APS and TEMED at the end. Remove bubbles. Fill the cassette completely with solution. Allow to polymerize at room temperature.
- 6. Set UP Gel Remove the comb carefully after gel has polymerized. Attach gel to the electrophoresis tank according the manufacturer’s instructions. Add catholyte (sodium hydroxide) to the upper buffer chamber and anolyte (Phosphoric acid) to the lower buffer chamber.
- 7. Sample Preparation & Loading Mix protein sample with equal volume of 2X loading buffer. Loading buffer includes the following reagents: Urea. Ampholyte solution pH 3.5 – 10. Ampholyte solution pH 4 – 6. Triton X-100. 2-Mercaptoethanol. 1% bromophenol blue. Distilled water. Centrifuge the sample to remove any aggregate. Apply the supernatant to the wells with a disposable tip or Hamilton syringe. Run the gel at 150V for 30 min and then at 200V for 2 hours.
- 8. Cutting slices forpH determination After electrophoresis, gel is cut into 0.5cm slices keeping track of the distance from any electrode. Place each slide into an eppendorf and label by the distance (in cm) from the anode. Incubate each slice in 1ml 10mM KCl for about 30 min. Centrifuge and read pH of the clear supernatant. A standard curve is obtained by plotting pH of the gel slices and the distance from the anode. Unknown pI can be determined.
- 9. Fixing and Stainingthe gel. Soak the gel in 10% TCA for the removal of ampholytes. Ampholyte removal is necessary to reduce background staining. Stain the gel with Coomassie blue for 10 min. Discard staining solution and replace with the destaining solution.
- 10. IEF in horizontalslab gels. Horizontal slab gel has several advantages. Cooling of the gel is efficient as the gel remains flat on the cooling plate. Lesser amount of electrode solutions are required. Gel size can be adjusted. Sample can be added at any position on pH gradient. Precast IEF gels are commercially available.
- 11. A Slab GelSystem for IEF.
- 12. 2D Gel Electrophoresis. An application of IEF. Protein separated in two dimensions. First on the base of pI. Can be performed in tubes of small diameter. Second on the basis of mol. Wt. in normal SDS PAGE. Procedure can be adapted by combining IEF and PAGE.
- 13.
Editor's Notes
- #3 Amphoteric molecules are those molecules who can have both positive and negative charge.