Heimgartner U, Kozulić B, Mosbach K
Department of Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland.
Anal Biochem. 1989 Aug 15;181(1):182-9. doi: 10.1016/0003-2697(89)90414-4.
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.
通过本研究中描述的四步程序，可以高选择性和高灵敏度地检测固定在膜上的糖蛋白。糖蛋白首先用高碘酸钠氧化，然后将聚丙烯酰基多肼与糖蛋白糖部分产生的醛基偶联。在第三步中，一种糖酶，如辣根过氧化物酶，通过其糖链中形成的醛基与聚合物上剩余的酰肼基团偶联。在最后一步中，通过结合的糖酶的反应产物实现糖蛋白的可视化。糖蛋白检测的灵敏度最关键地取决于酰肼试剂。因此，二酰肼不太令人满意，三酰肼较好，而多酰肼是最好的。使用了两种不同的多酰肼。一种基于丙烯酰胺，另一种基于N-丙烯酰基-三（羟甲基）氨基甲烷。第二种被证明更优越，因为它具有更高的灵敏度且没有可检测到的背景染色。我们还研究了各种反应条件对具有低聚甘露糖和N-乙酰乳糖胺型糖链的糖蛋白染色的影响。其中一些，如转化酶和胎球蛋白，可以以类似于凝胶中的银染和膜上的胶体金染的灵敏度进行检测。只有当几个N-乙酰葡糖胺残基仍与蛋白质结合时，在标准条件下才可能检测到少量内切糖苷酶H去糖基化的糖蛋白。
