Theory:
Isoelectric focusing(IEF), performing a very important role in protein separation technology, is utilized to separate amphoteric macromolecules in line with the different isoelectric points.
Filling the electric field with amphiphilic carriers and anti-convection media. When the electric field is applied, a stable pH gradient between the poles will be established as a result of the movement of amphiphilic carriers. Protein molecules or other amphiphilic molecules in such pH gradient will reach a zone with zero static charge due to the movement of their surface charge in this electric field. And then the pH can be regarded as the pI of this molecule. Simultaneously, molecules focusing on isoelectric points will also spread. And because of the change of pH, molecules will get a positive or negative charge, as long as they are away from their isoelectric points. And then it migrats to pI.
Instruments and reagents
Materials: proetin samples
Reagents:
Polyacrylamide, B polyacrylamide, amphoteric electrolytes, urea, NP-40, teiton-100
Electrode solution: 1M phosphoric acid (anolyte), 1M sodium hydroxide (catholyte)
Fixative: 100g TCA, 10g sulfosalicylic acid dissolved in 500ml, a total volume of 1000ml
Staining solution: 0.35g Coomassie brilliant blue R-150 was dissolved in 300ml destaining solution and heated to 60-70 ℃, copper sulfate was added 0.3g
Destaining solution: 25% ethanol, 8% acetic acid is dissolved in water
Sample Buffer: 1% Ampholine, 2% Triton X-100,9M urea
Steps:
1. Sample preparation:
Extracting pending samples with IEF sample buffer.
2. Mould
Performing two clean special IEF glass with suicide and anti-suicide reaction. And then clamping the glass sheets with good position.
3. Preparing glue
Glue Composition: 6ml gum stock solution (10%, 19/1), 6-8% urea, 1ml Ampholine (pH3.5-10), 60-80ul 10% AP and 5 ul TEMED.
4. Pouring glue: pouring glue into mould.
5. Electrophoresis:
Uncovering the up and down glass sheets after the gel coagulation. Placing the plastic gasket with clean bottom on the electrophoresis tank. And then putting an electrode strip impregnated with electrode buffer on both sides of plastic surface. Placing a lens mirror paper in any position on the surface of glue. And loading samples on paper. Closing the lid, and performing electrophoresis with 25W. After 10 minutes, removing paper and stopping electrophoresis. And then continuing electrophoresis for 30 minutes. And then stop it once the electric current reaches 4mA.
6. Determination of isoelectric point of protein
7. Fix: remove the plastic sheet, placing into fixative for 15min.
8. Staining: remove the plastic pieces, place into the 65 ℃ dye solution until bands appear.
9. Storing it dry after eliminating background.
Isoelectric focusing is widely applied in the determination of isoelectric points of protein, as well as protein separation.
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determination of isoelectric point of protein