Capillary Sodium Dodecyl Sulfate Research Articles

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of θ-amino acid buffers (pH≈5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an ϵ-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 μm I.D. capillary or narrow bore capillaries without a polymer solution (25 μm I.D.) were employed. rtPA was resolved into at least eight species within a p I range of 6.4–9.2 using Ampholine 3.5–10. Migration time precision for the major peaks ranged from 0.2% for CZE to ≤2–3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

Sodium dodecyl sulfate capillary electrophoresis of proteins in entangled solutions of poly(vinyl alcohol)

A novel polymer network is described for efficient sieving of sodium dodecyl sulfate (SDS)-protein complexes: poly(vinyl alcohol) (PVA; average M r 133 000). The entanglement threshold of these solutions was found to be at 3% (w/v) PVA. Solutions from 4 to 6% PVA offer excellent resolution in the 14 400-94 000 protein molecular mass interval. Ferguson plot analysis showed that the separation is indeed based on mass discrimination, as it should in SDS electrophoresis, with extrapolated (at 10% polymer) limit values of mobility for all particles in the range (2.34-2.87) · 10 −8 m 2 V −1s −1. The advantages of PVA are full transparency in the UV region down to 200 nm and extremely low viscosities (e.g., a 5% PVA solution has a viscosity 25 times greater than that of buffer at 30°C). A unique wall effect was found, by which, on decreasing the inner diameter of the capillary from 75 to 25 μm, the apparent entanglement threshold was shifted to extremely dilute PVA solutions, since in 25-μm capillaries efficient sieving was obtained below 1% PVA, i.e., at concentrations well below the entangled regime. It is hypothesized that residual, free silanols present (even in a coated capillary) act as nucleation sites for H-bond formation and aggregation of free PVA molecules.

Ultra-fast high-performance capillary sodium dodecyl sulfate gel electrophoresis of proteins

An ultra-fast analysis of proteins, based on sodium dodecyl sulfate (SDS)-mediated gel electrophoresis was developed, in which protein molecular mass standards ranging from M r 14 200 to 94 700 were separated within 3 min. A 50 μm diameter uncoated fused-silica capillary column and a high field strength are used. The effects of the SDS concentration in the separation gel buffer and in the sample buffer on the resolution of protein test mixture were studied. The influence of the heat treatment of the sample prior analysis is also discussed.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins II. On the Ferguson method in polyethylene oxide gels

Capillary sodium dodecyl sulfate (SDS) gel electrophoresis is demonstrated to be a powerful new analytical method for the separation of protein molecules based on their molecular mass. Standard curves of logarithm molecular mass versus reciprocal relative migration time give the estimated molecular mass of sample proteins within the acceptable 10% error for most proteins. Larger errors are possible, however, when special groups such as carbohydrates (glycoproteins) or lipids (lipoproteins) are present due to a different ratio of binding SDS molecules. An automated Ferguson method is shown that corrects for this non-ideal behavior in capillary SDS-polyethylene oxide gel electrophoresis.

High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymeized in fused-silica capillaries of 75 μm I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40 000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer compositin (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.