Gillespie P G, Gillespie S K
Department of Physiology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Anal Biochem. 1997 Mar 15;246(2):239-45. doi: 10.1006/abio.1997.2019.
During electrophoresis and electroblotting to transfer membranes, picogram amounts of protein can react irreversibly with the polyacrylamide matrix, preventing complete electrophoresis and efficient electroblotting. Bovine hemoglobin, but not other potential carrier proteins, mitigates this protein loss by migrating with or ahead of other proteins and scavenging reactive groups. Inclusion of 5 micrograms of hemoglobin in sample wells increases by 4-fold the amount of a radiolabeled test protein, myosin I beta, found at its appropriate 120-kDa position in sodium dodecyl sulfate-polyacrylamide gels. For electroblotting, incubating the gel with 0.25 mg/ml hemoglobin prior to transfer improves mobilization of picogram amounts of radiolabeled myosin I beta out of the gel by about 6-fold. For picogram amounts of proteins, therefore, approximately 20-fold more protein transfers to a blotting membrane when hemoglobin is used during both electrophoresis and transfer. This effect is general: transfer of radiolabeled Drosophila embryo proteins is improved dramatically by including hemoglobin in the pretransfer incubation solution. We suggest that electroblot-based detection of small amounts of protein, particularly when in the absence of other potential carrier proteins, can be improved substantially by using hemoglobin.
在进行电泳和电转印至膜的过程中，皮克级量的蛋白质会与聚丙烯酰胺基质发生不可逆反应，从而阻止完全电泳和高效电转印。牛血红蛋白而非其他潜在的载体蛋白，通过与其他蛋白质一起迁移或先于其他蛋白质迁移并清除反应性基团，减轻了这种蛋白质损失。在样品孔中加入5微克血红蛋白，可使在十二烷基硫酸钠-聚丙烯酰胺凝胶中处于合适的120 kDa位置的放射性标记测试蛋白β-肌球蛋白的量增加4倍。对于电转印，在转移前将凝胶与0.25 mg/ml血红蛋白一起孵育，可使皮克级量的放射性标记β-肌球蛋白从凝胶中迁移出来的效率提高约6倍。因此，对于皮克级量的蛋白质，在电泳和转移过程中使用血红蛋白时，转移到印迹膜上的蛋白质大约多20倍。这种效果具有普遍性：在转移前孵育溶液中加入血红蛋白可显著提高放射性标记的果蝇胚胎蛋白的转移效率。我们建议，通过使用血红蛋白，基于电转印的少量蛋白质检测，尤其是在没有其他潜在载体蛋白的情况下，可得到显著改善。
