实验方案

工作溶液配制

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

1. 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。
2. 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。 

1. 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。
2. 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. 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。

参考文献

Identification of a Small Probe That Can Be Conjugated to Proteins by Proximity Labeling.
Authors: Sun, Weiping and Huo, Yinbo and Mei, Yuxuan and Zhou, Qingtong and Zhao, Suwen and Zhuang, Min
Journal: ACS chemical biology (2020): 39-43

Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection.
Authors: Noviana, Eka and Jain, Sidhartha and Hofstetter, Josephine and Geiss, Brian J and Dandy, David S and Henry, Charles S
Journal: Analytical and bioanalytical chemistry (2020): 3051-3061

A neuraminidase potency assay for quantitative assessment of neuraminidase in influenza vaccines.
Authors: Byrne-Nash, Rose T and Gillis, Jacob H and Miller, David F and Bueter, Katie M and Kuck, Laura R and Rowlen, Kathy L
Journal: NPJ vaccines (2019): 3

Author Correction: A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction.
Authors: Iliopoulou, Maro and Nolan, Rory and Alvarez, Luis and Watanabe, Yasunori and Coomer, Charles A and Jakobsdottir, G Maria and Bowden, Thomas A and Padilla-Parra, Sergi
Journal: Nature structural & molecular biology (2019): 526

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging.
Authors: Alam, Md Kausar and El-Sayed, Ayman and Barreto, Kris and Bernhard, Wendy and Fonge, Humphrey and Geyer, C Ronald
Journal: Molecular imaging and biology (2019): 54-66

Highly efficient electrochemical sensing platform for sensitive detection DNA methylation, and methyltransferase activity based on Ag NPs decorated carbon nanocubes.
Authors: Gao, Fenglei and Fan, Taotao and Ou, Shanshan and Wu, Jing and Zhang, Xing and Luo, Jianjun and Li, Na and Yao, Yao and Mou, Yingfeng and Liao, Xianjiu and Geng, Deqin
Journal: Biosensors & bioelectronics (2018): 201-208

Improved performance of lateral flow immunoassays for alpha-fetoprotein and vanillin by using silica shell-stabilized gold nanoparticles.
Authors: Lu, Xuewen and Mei, Ting and Guo, Qi and Zhou, Wenjing and Li, Xiaomei and Chen, Jitao and Zhou, Xinke and Sun, Ning and Fang, Zhiyuan
Journal: Mikrochimica acta (2018): 2

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.
Authors: Brown, Koshonna and Thurn, Ted and Xin, Lun and Liu, William and Bazak, Remon and Chen, Si and Lai, Barry and Vogt, Stefan and Jacobsen, Chris and Paunesku, Tatjana and Woloschak, Gayle E
Journal: Nano research (2018): 464-476

Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.
Authors: Stephen, Laurie and Schwarz, Emanuel and Guest, Paul C
Journal: Advances in experimental medicine and biology (2017): 149-156

Multiplex Immunoassay Profiling.
Authors: Stephen, Laurie
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 169-176

实验方案

工作溶液配制

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

1. 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。
2. 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。 

1. 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。
2. 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. 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。然后将细胞与小鼠抗微管蛋白抗体一起孵育，并用ReadiLink™xtra Rapid iFluor™647抗体标记试剂盒（#1963）标记的山羊抗小鼠IgG染色。

参考文献

Identification of a Small Probe That Can Be Conjugated to Proteins by Proximity Labeling.
Authors: Sun, Weiping and Huo, Yinbo and Mei, Yuxuan and Zhou, Qingtong and Zhao, Suwen and Zhuang, Min
Journal: ACS chemical biology (2020): 39-43

Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection.
Authors: Noviana, Eka and Jain, Sidhartha and Hofstetter, Josephine and Geiss, Brian J and Dandy, David S and Henry, Charles S
Journal: Analytical and bioanalytical chemistry (2020): 3051-3061

A neuraminidase potency assay for quantitative assessment of neuraminidase in influenza vaccines.
Authors: Byrne-Nash, Rose T and Gillis, Jacob H and Miller, David F and Bueter, Katie M and Kuck, Laura R and Rowlen, Kathy L
Journal: NPJ vaccines (2019): 3

Author Correction: A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction.
Authors: Iliopoulou, Maro and Nolan, Rory and Alvarez, Luis and Watanabe, Yasunori and Coomer, Charles A and Jakobsdottir, G Maria and Bowden, Thomas A and Padilla-Parra, Sergi
Journal: Nature structural & molecular biology (2019): 526

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging.
Authors: Alam, Md Kausar and El-Sayed, Ayman and Barreto, Kris and Bernhard, Wendy and Fonge, Humphrey and Geyer, C Ronald
Journal: Molecular imaging and biology (2019): 54-66

Highly efficient electrochemical sensing platform for sensitive detection DNA methylation, and methyltransferase activity based on Ag NPs decorated carbon nanocubes.
Authors: Gao, Fenglei and Fan, Taotao and Ou, Shanshan and Wu, Jing and Zhang, Xing and Luo, Jianjun and Li, Na and Yao, Yao and Mou, Yingfeng and Liao, Xianjiu and Geng, Deqin
Journal: Biosensors & bioelectronics (2018): 201-208

Improved performance of lateral flow immunoassays for alpha-fetoprotein and vanillin by using silica shell-stabilized gold nanoparticles.
Authors: Lu, Xuewen and Mei, Ting and Guo, Qi and Zhou, Wenjing and Li, Xiaomei and Chen, Jitao and Zhou, Xinke and Sun, Ning and Fang, Zhiyuan
Journal: Mikrochimica acta (2018): 2

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.
Authors: Brown, Koshonna and Thurn, Ted and Xin, Lun and Liu, William and Bazak, Remon and Chen, Si and Lai, Barry and Vogt, Stefan and Jacobsen, Chris and Paunesku, Tatjana and Woloschak, Gayle E
Journal: Nano research (2018): 464-476

Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.
Authors: Stephen, Laurie and Schwarz, Emanuel and Guest, Paul C
Journal: Advances in experimental medicine and biology (2017): 149-156

Multiplex Immunoassay Profiling.
Authors: Stephen, Laurie
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 169-176