Serum protein fractionation using supported molecular matrix electrophoresis

Abstract

Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

A fixation method for the optimisation of western blotting

“…One fixation method which used 0.5% (v/v) glutaraldehyde for the detection of low molecular-weight acidic and basic isoelectric point (pI) proteins increased the sensitivity of western blotting from 1.5- to 12-fold 12 . Additionally, a new electrophoresis method, supported molecular matrix electrophoresis (SMME), has been developed using PVDF membranes, which does not require any transfer, but uses the fixation step for immunoblotting, applying acetone immersion and/or heating of the membrane after electrophoresis 13,14 . Organic solvents such as acetone and methanol are often used as fixatives for the immunostaining of tissues or cells 15 .…”

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans

“…As shown in previous reports, N-linked glycans can be released from glycoproteins by heating to 80–95°C under alkaline conditions [ 19 , 20 ]. Chemical de-N-glycosylation is considered alkaline hydrolysis of the glycosyl carboxamide of Asn.…”

“…Reductive beta elimination using a dilute base in the presence of sodium borohydride has often been used to release O-linked glycans from glycoproteins. N-linked glycans are also detected as minor products in the glycan mixture obtained from this reaction [ 19 , 20 ]. O-linked glycans are reduced into the corresponding alditols in the reaction, whereas N-linked glycans are detected in their unreduced form.…”