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
图1.将 HL-60细胞与（红色）或没有（绿色）抗人HLA-ABC（W6 / 32 mAb）一起孵育。然后将细胞与使用ReadiLink™xtra Rapid iFluor™750抗体标记试剂盒（#1965）标记的山羊抗小鼠IgG一起孵育。使用APC-Cy7通道中的ACEA NovoCyte流式细胞仪检测荧光信号。
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