Direct recovery of proteins into a free-liquid phase after preparative isoelectric focusing in immobilized pH gradients.
A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The transfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: short electrophoretic times; avoidance of anodic oxidation; chilling of the buffer in the anodic chamber; and use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).
D004798:Enzymes / Q000302:isolation & purification; D006441:Hemoglobin A / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D012995:Solubility
D004798:Enzymes; D011506:Proteins; D006441:Hemoglobin A