ISOELECTRIC FOCUSINGM. KALEEM IQBAL0309-MPHIL-BIO-T-12Institute of Industrial BiotechnologyGCU, Lahore.
Isoelectric FocusingProcess by 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.
Amphoteric molecules are those molecules who can have both positive and negative charge. 2Iso-electric Point (pI)The pH 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.
AmpholytesEstablishment of stable 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.
Working Procedure. Following Chemicals 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.
Set UP GelRemove the comb carefully after gel has polymerized. Attach gel to the electrophoresis tank according the manufacturers instructions. Add catholyte (sodium hydroxide) to the upper buffer chamber and anolyte (Phosphoric acid) to the lower buffer chamber. Sample Preparation & LoadingMix 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. Cutting slices for pH determinationAfter 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.
Fixing and Staining the 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.
IEF in horizontal slab 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. A Slab Gel System for IEF.
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.