Urs Heimgartner

Polyacrylic polyhydrazides as reagents for detection of glycoproteins

Glycoproteins immobilized on membranes can be detected with high selectivity and sensitivity by the four-step procedure described in this work. The glycoproteins are first oxidized by sodium periodate and then polyacrylic polyhydrazides are coupled to the aldehyde groups generated in the sugar part of the glycoproteins. In the third step, a glycoenzyme, such as horseradish peroxidase, is coupled to the remaining hydrazide groups on the polymer through the aldehydes formed in its glycan chains. In the last step, the visualization of glycoproteins is achieved through the reaction product of the bound glycoenzyme. The sensitivity of the glycoprotein detection is most critically dependent on the hydrazide reagent. Thus, dihydrazides were not satisfactory, a trihydrazide was better, and polyhydrazides were the best. Two different polyhydrazides were used. One was based on acrylamide and the other on N-acryloyl-tris(hydroxymethyl)aminomethane. The second one proved to be superior because it gave higher sensitivity with no detectable background staining. We have also investigated the influence of various reaction conditions on staining of glycoproteins having oligomannose and N-acetyllactosamine type glycan chains. Some of them, invertase and fetuin, could be detected with sensitivity similar to that of silver staining in gels and colloidal gold staining on the membranes. The detection of small quantities of Endo H-deglycosylated glycoproteins was possible under standard conditions only if several N-acetylglucosamine residues remained bound to the protein.

An apparatus for submerged gel electrophoresis

A novel apparatus for submerged gel electrophoresis is described in detail. It includes an upper buffer compartment, a lower buffer compartment, and a horizontal plate between the two compartments. The horizontal plate is a heat exchanger connected to an external heater/cooler. Buffer circulates between the two compartments through openings in the horizontal plate. In the upper compartment two separated openings are positioned on each side of the horizontal plate between the side walls and long vertical barriers. The barriers initially direct the flow of buffer and define the electric field on the sides of the upper compartment. The electric field is confined essentially into a rectangular box, defined on the ends by the end walls, on the sides by the barriers, on the bottom by the cooling plate, and on the top by the air. Since the volume of buffer is smaller in the electrode compartment than in the reservoir under the cooling plate, this design enables formation of a substantially uniform electric field without creating too high a current. To enhance uniformity of the electric field, anode and cathode consist each of two platinum wires positioned one above the other at a distance of 6 mm. The electrodes can be placed parallel to the sides and perpendicular to the buffer flow or parallel to the ends and the flow of buffer. The stream of buffer in the upper compartment is regulated by two dams, perpendicular to the long barriers, on each end of the horizontal plate.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyacrylic polyhydrazides as novel reagents for detection of antibodies in immunoblotting assays

By the glycoprotein specific staining method introduced recently (Heimgartner et al., 1989, Anal. Biochem. 181, 182-189) it is also possible to detect an antibody bound to its antigen on a membrane. The antibody is oxidized by periodate prior to incubation. Next, a polyacrylic polyhydrazide is coupled to the aldehyde groups generated in the sugar part of the antibody molecule. A periodate oxidized glycoenzyme, such as horseradish peroxidase, is then coupled to the remaining hydrazide groups of the polymer and incubation with a suitable enzyme substrate visualizes the glycoenzyme-polyhydrazide-antibody-antigen complex. The sensitivity of the detection is most critically dependent on the antibody class and the polyhydrazide reagent. Oxidation conditions are less important and the antibody does not need to be purified prior to periodate oxidation. Under standard conditions, the sensitivity obtained with IgG type antibodies was about ten times lower than with peroxidase-labeled secondary antibodies. However, a similar if not higher sensitivity can be expected with more glycosylated antibodies, such as IgM or IgE, or with chicken antibodies. This approach is advantageous because antibodies of all classes and from all species can be detected with the same reagent, the polyhydrazide, no foreign molecule is introduced in the antibody before its binding to the antigen and no conjugate needs to be prepared in advance.

Hydrophilic and Amphiphatic Monomers and use of their Gels as Separation Media

Abstract In this contribution we describe some results of the work carried out by Professor Klaus Mosbachs group at the Swiss Federal Institute of Technology and later at Elchrom. Synthesis of new acrylic monomers was required for the protein imprinting project and, after the finding that N -acryloyl-tris(hydroxymethyl)aminomethane gels offer advantages for electrophoresis, several series of monomers were synthesized and used to prepare separation media. Particularly useful were hydrophilic monomers based on amino sugar alcohols and corresponding amphiphatic monomers that enable separation of proteins by hydrophobic interaction electrophoresis.