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虫号: 2084389
注册: 2012-10-24
专业: 电化学分析

[求助] Ru标记抗体的问题

求助

用自己制备的RuNHS按照文献的方法与抗体结合，用葡聚糖凝胶柱洗脱后理论上先出来的是抗体，但是在400-600nm处不出峰，所以不知道抗体和Ru连上了没有。跪求做过该类实验的小伙伴们，能告知详细的操作方法。、
我参考的文献：Ru tris(2,2'-bipyridine-4,4'-dicarboxylic acid) N-hydroxysuccinimide ester
(4). 0.46 g of DCC and 0.238 g of N-hydroxysuccinimide were dissolved
in 3 ml of DMF with stirring and cooled in an ice bath. A solution of 0.38
g of Ru tris(2,2'-bipyridine-4,4'-dicarboxylic acid) (2) was added, and the
mixture was stirred for a few hours. The formed precipitate was removed
by filtration through a syringe filter, and the filtrate containing the active
Ru-complex was used for labeling the substrates. The NHS-ester (3) was
prepared in analogy to (4).
The proteins HSA, IgG, ConA, and Ferritin were obtained from Sigma
Chemical Co. (St. Louis, MO) and used without further purification. The
proteins (10 mg portions) were labeled by adding a 100-fold molar excess
of the Ru-NHS ester in 50 jLI of DMF to 1 ml of stirred protein solution
(0.2 M carbonate buffer, pH 8.3-9.1), followed by a 2-6 h incubation and
purification of the labeled protein by gel filtration chromatography on
Sephadex G-25 or G-50, using 0.1 M PBS, pH 7.2.

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