[Capillary isoelectric focusing of protein]

A capillary isoelectric focusing (CIEF) method was developed for the separation of proteins with different pI. Uncoated capillary (57 cm x 50 microm i.d.) was used instead of commercial coated column because of its ease in use and a longer lifetime. Polymeric additive, methylcellulose (MC-1500), acts as dynamic coating of the silica wall to suppress electroendosmosis. TEMED was used to extend the pH range of ampholine. Taurine was added to the ampholyte mixture to eliminate the aggregation of proteins. The uniform design, a new experimental design method suited for experiments of a large number of factors and levels, was used for the optimization of experimental conditions, such as concentrations of ampholine, MC-1500, TEMED and Taurine. U6* (6(4)) table was chosen to arrange experiments. Regression equations of maximum current, mean migration time and criterion of resolution power were obtained after the treatment of experimental results by SAS software. Optimized experimental conditions were obtained according to multivariate regression equations. Carrier ampholyte which consists of 3.2%-4.8% ampholine 3-10, 0.06%-0.18% MC-1500, 0.8% TEMED and 1%-2% taurine will produce good resolution. Cathode and anode solutions were 20 mmol/L NaOH and 20 mmol/L HsPO4, respectively. Low pressure (34.5kPa) was applied at the inlet of the capillary simultaneously after 31 minutes of isoelectric focusing at 25 kV to mobilize the focused acidic protein zone to pass through the detector. Human serum albumin (HSA) and human epidermal growth factor (hEGF) were separated by this method using an ampholyte mixture of 4% ampholine 3-10, 0.8% TEMED, 1% taurine and 0.12% MC-1500. Data analysis indicated that the coefficients of variation of pressure and the method are all 1.2%. The results demonstrated that the method established is applicable to basic, neutral and acidic proteins, and can be used for the determination of pI by internal calibration.