Abelev G I, Karamova E R, Lazarevich N L, Kiseleva V I, Poverennyĭ A M
Mol Biol (Mosk). 1994 Jul-Aug;28(4):768-77.
A very strong electroosmotic counterflow was produced on nitrocellulose membranes during isotachophoresis in a system of 0.06 M Tris-HCl (pH 6.7) as the leading electrolyte and 0.012 M Tris-beta-alanine (pII 8.6) as the terminating one. This counterflow was equal in rate and opposite in direction to the migration of the Cl-/beta-alanine boundary. The rate of counterflow was much higher than the rate of migration of any organic anions, including different proteins. Double-stranded and single-stranded DNA or its adducts were fixed on the nitrocellulose membrane, and the membrane was blocked with unrelated proteins. DNA-binding proteins, namely antibodies to DNA, followed by peroxidase-conjugated anti-IgG, were introduced into the counterflow, which transferred them one after another to the DNA spots. Thus, sequential binding and washing was performed automatically. In this way, antibodies were detected to ds- and ss-DNA, to BrdU-DNA, to Z-DNA, to biotinylated DNA and DNA modified with trans-Pt, as well as development of biotinylated DNA dots by steptavidin-peroxidase.
在等速电泳过程中，以0.06 M Tris-HCl（pH 6.7）作为前导电解质、0.012 M Tris-β-丙氨酸（pH 8.6）作为终止电解质的体系中，硝酸纤维素膜上产生了非常强的电渗逆流。这种逆流在速率上与Cl⁻/β-丙氨酸边界的迁移相等，方向相反。逆流速率远高于任何有机阴离子（包括不同蛋白质）的迁移速率。将双链和单链DNA或其加合物固定在硝酸纤维素膜上，并用无关蛋白质封闭该膜。将DNA结合蛋白，即抗DNA抗体，随后加入过氧化物酶偶联的抗IgG，引入逆流中，逆流将它们逐个转移到DNA斑点上。因此，自动进行了顺序结合和洗涤。通过这种方式，检测到了针对双链和单链DNA、BrdU-DNA、Z-DNA、生物素化DNA和经反式铂修饰的DNA的抗体，以及通过链霉亲和素-过氧化物酶对生物素化DNA点进行显色。
