Lutomski D, Caron M, Cornillot J D, Bourin P, Dupuy C, Pontet M, Bladier D, Joubert-Caron R
Laboratoire de Biochimie et Technologie des Protéines, Université Paris-Nord, UFR Léonard de Vinci, Bobigny, France.
Electrophoresis. 1996 Mar;17(3):600-6. doi: 10.1002/elps.1150170331.
Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.
脊椎动物可溶性β-半乳糖苷结合凝集素构成了一个不断扩大的蛋白质家族，最近被命名为半乳糖凝集素。在哺乳动物中，已对七种不同的半乳糖凝集素进行了测序和表征，并且有确凿证据表明该凝集素家族还存在其他成员。六种半乳糖凝集素中的三种是同型二聚体，（i）具有相对分子质量约为14500的相同亚基，（ii）氨基酸序列同源性导致可能的免疫化学交叉反应性。通过传统的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），即使随后进行免疫印迹，它们彼此也无法区分。然而，它们不同的等电点使得可以使用等电聚焦和二维（2-D）聚丙烯酰胺凝胶电泳来鉴定它们。基于对纯化的半乳糖凝集素的特定斑点和粗提物斑点进行固定化pH梯度二维电泳（IPG-Dalt）分析，开发了一种在细胞和组织的粗提物中鉴定这些半乳糖凝集素的策略，分析后进行银染。此外，使用抗半乳糖凝集素1（Gal-1）和抗碳水化合物结合蛋白15（CPB15）抗体对脑和白血病细胞（HL60）进行二维免疫印迹，从而鉴定相关多肽。我们的结果表明，使用IPG-Dalt具有良好的重现性，即使在碱性等电点下也能检测到半乳糖凝集素或其他半乳糖苷结合蛋白。
