Abstract: In a method for immobilizing biomolecules and affinity ligands water insoluble matrices, having amino groups and selected from test tubes, microtiter plates, microscope slides, beads, membranes, resins, and filters, are reacted with a cyclobutene carboxylic acid derivative, such as cyclobutene carboxylic acid diester, cyclobutene carboxylic acid halide, cyclobutene carboxylic acid ester halide, cyclobutene carboxylic acid dialkoxyester, and cyclobutene carboxylic acid imidazole, as an activating compound in methanol and triethylamine to form active matrices with active groups. A protein containing at least one primary or secondary amino group, is dissolved and added to the matrices. The activated matrices and the dissolved protein are incubated at pH of 7-10 and a temperature of +4° C. to +60° C. in an aqueous buffer system, free of primary and secondary amines, to thereby immobilize the protein on the matrices.