实验方案
工作溶液配制
蛋白质工作溶液(A)
为了标记50 µg蛋白质(假设目标蛋白质浓度为1 mg / mL),请将5 µL(反应总体积的10%)反应缓冲液(组分B)与50 µL目标蛋白质溶液混合。
注意:如果蛋白质浓度不同,请相应地调整蛋白质体积,以使〜50 µg蛋白质可用于标记反应。
注意:要标记100 µg蛋白质(假设目标蛋白质浓度为1 mg / mL),请将10 µL(反应总体积的10%)反应缓冲液(组分B)与100 µL目标蛋白质溶液混合。
注意:蛋白质应溶于1X磷酸盐缓冲盐水(PBS),pH 7.2-7.4;如果蛋白质溶解在甘氨酸缓冲液中,则必须针对1X PBS(pH 7.2-7.4)进行透析,或使用10 kDa的Amicon Ultra-0.5,Ultracel-10膜去除游离的胺或铵盐(例如硫酸铵和乙酸铵)被广泛用于蛋白质沉淀。
注意:为获得最佳标记效率,建议最终蛋白质浓度范围为1-2 mg / mL,结合效率明显降低,低于1 mg / mL。
操作步骤
运行缀合反应
- 将蛋白质工作溶液(溶液A)添加到一个小瓶的标记染料(组分A)中,并通过将小瓶涡旋几秒钟将它们充分混合。
注意:如果要标记100 µg的蛋白质,请使用两个小瓶(组分A),将100 µg的蛋白质分成2 x 50 µg的蛋白质,并使每个50 µg的蛋白质与一小瓶的标记染料反应。然后合并两个小瓶,用于下一步。
- 将缀合反应混合物在室温下放置30-60分钟。
注意:如果需要,可以旋转或摇动缀合反应混合物更长的时间。
停止缀合反应
- 将5 µL(对于50 µg蛋白质)或10 µL(对于100 µg蛋白质)添加到缀合反应混合物中,占TQ™染色猝灭缓冲液(组分C)总反应体积的10%;混合均匀。
- 在室温下孵育10分钟。标记的蛋白(抗体)可以使用了。
蛋白质缀合物的储存
蛋白质缀合物应在载体蛋白(例如0.1%牛血清白蛋白)存在下以> 0.5 mg / mL的浓度存储。为了更长的存储时间,可以将蛋白质缀合物冻干或分成单份使用,并存储在≤–20°C下。
图示
![ReadiLink™xtra Rapid iFluor™350抗体标记试剂盒(标记50ug抗体) 货号1950](http://www.gewhatman.cn/wp-content/uploads/2022/05/20220509_627902a2007fa.jpg)
图1. HeLa细胞中微管蛋白的免疫荧光染色。HeLa细胞用4%PFA固定,用0.1%Triton X-100透化并封闭。然后将细胞与兔抗微管蛋白单克隆抗体一起孵育,并用ReadiLink™xtra Rapid iFluor™350抗体标记试剂盒(#1950)标记的山羊抗兔IgG染色。
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