An improved method of high‐voltage isoelectric focusing on cellulose acetate membranes

Abstract

An apparatus and the procedure for cellulose acetate isoelectric focusing were refined and optimized for routine use in clinical laboratories. The space in the electrophoretic chamber was minimized to maintain high humidity during the run. The water content of the thin cellulose acetate membranes in the chamber was well controlled during isoelectric focusing and the effect of atmospheric carbon dioxide was also excluded. The temperature of the membranes was kept below 2 degrees C, even under conditions of high electric field strength for faster isoelectric focusing of high-molecular-weight proteins. No special training was required for technicians in clinical laboratories to obtain reproducible isoelectric profiles of human serum proteins.

New detection method after cellulose acetate membrane isoelectric focusing: Activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple Western blotting

“…The conditions for IEF, CBB staining, transferrin detection, and analysis of electrophoretic pattern were carried out according to our method described (1,4,5).…”

“…In contrast, the cellulose acetate (CA) membrane is easy to handle and frequently used as a support medium in many clinical laboratories. Using the CA membrane as a supporting medium, we previously developed an IEF apparatus and reported optimal conditions for clearly detecting serum proteins as well as glycoproteins and specific proteins (1)(2)(3)(4).…”

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing

“…After electrophoresis, the six sheets of CA membrane were peeled off one by one. The top CA membrane was stained with Coomassie brilliant blue G-250 according to the method of Toda et al (8).…”

“…There have been some reports of the analysis of the M protein using IEF (5)(6)(7), but there are no reports yet on the simultaneous detection of different immunoglobulins. We developed a method for IEF using cellulose acetate membrane (CA membrane), and previously reported that it could detect microheterogeneity of five different immunoglobulins (IgM, IgA, IgG, κ-chain, and γ-chain) and a protein fraction simultaneously (8)(9)(10). In this study, we attempted to examine differences in the isoelectric pattern of each immunoglobulin, in the pI of M protein, and in the M protein binding sugar chain between MM and MGUS patients using this IEF method.…”

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes

“…We have developed an apparatus for isoelectric focusing using cellulose acetate (CA) membranes, and optimized the procedures (1,2). Isoelectric focusing has been shown to be a useful technique for protein analysis.…”