【Promega】HiBiT 蛋白标签系统介绍

【Promega】HiBiT 蛋白标签系统介绍

HiBiIT 蛋白标签系统

将生物发光应用于蛋白分析


使用 HiBIT 蛋白标签系统,可开发出操作简便、灵敏度高的蛋白定量方法,来检测蛋白丰度或定位的变化。HiBiT 系统简化了活细胞中的蛋白标签,提供了一种精简、无抗体检测方案,仅需使用光度计进行信号定量。HiBiT 技术凭借其测定内源性表达水平灵敏度高和仅需加入单一试剂的便利,为蛋白生物学研究人员开辟了无限可能。

原理


HiBiT 是一个由 11 个氨基酸构成的肽标签,可以连接到任何目的蛋白(POI),可在10分钟或更短时间内使用生物发光试剂进行检测。检测试剂含有一个无活性的萤光素酶亚基 LgBiT,其能快速与 HiBIT结合,产生一种高活性萤光素酶,


用于内源性生物学的高灵敏蛋白定量技术

较宽的线性动态范围可准确定量 HiBiT 标签蛋白,不受蛋白表达水平的影响。HiBiT 检测系统的检测限<10-19摩尔,支持在内源性表达水平对低丰度蛋白进行定量。

【参考文献】

1.Schwinn, M.k. et al. (2020) A simple and scalable strategy for analysis of endogenous protein dynamics. PSc Rep. 10(1):8953

2.Boursier, M.E. et a. (2020) The luminescent HiBiT peptide enables selective quantitation of G protein-coupled ligandenaagementand internalization in livina cells.y, Biol. Chem. 295(15):5124-5135

3.Riching, K.M. et al. (2018) Quantitative Live-Cell Kinetic Degradation and Mechanistic Profiling of PROTAC Mode of Action.ACS Chem Biol.13(9):2758-2770

4.Sasaki, M. et a/. (2018) Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay. Virus Res. 243:69-79

5.Schwinn, M.k. et a. (2018): CRlSPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide AcS chem Bio.13(2):467-474

6.Oh-Hashi, K. ef al. (2017) Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells Biochem Biophys Rep.12:40-45

7.Rouault, A. A. J. et al. (2017) Regions of MRAP2 reguired for the inhibition of orexin and prokineticin receptor signaling Biochim Biophys Acta.1864:2322-2329


HiBiT 标签与定量工作流程


HiBiT 标签蛋白的定量:HiBiT 标签蛋白定量仅需“加入 - 读数”测定,无需抗体,操作简单。(A)通过 LgBiT亚基的瞬时或稳定共表达与其中一种非裂解性 Nano-Glo® 底物,实时动力学研究可达 72 小时。(B)裂解检测试剂定量 HiBiT 标签蛋白的总量,而细胞外检测试剂检测细胞表面或分泌型的标签蛋白的量。(C)通过使用 HiBiT 印迹试剂,在常规蛋白免疫印迹分析膜上快速测定 HiBiT 标签蛋白的大小。

通过内参报告基因归一化来控制特异性

HiBiT NanoDLR™

Nano-Glo®HiBiT 双萤光素酶®报告基因检测系统可以在同一孔中对 HiBiT 标签蛋白和萤火虫萤光素酶(Fluc)进行多重检测。该检测系统可最大程度减少或消除由转染效率、细胞数量、细胞活性、温度或检测时间等因素导致的实验变异性,从而提高数据质量。检测 HiBiT目的蛋白(POI)的调控降解时,该方法可以区分蛋白表达水平总体变化导致的特异性效应。

POI的编码序列(coding sequence ofthe POl, GOl)克隆到一个带有N端或C端 HiBiT 标签的多克隆位点(MCS)。HiBIT 融合蛋白的终止密码子之后是一个IRES序列,该序列能够使来自相同 mRNA的非融合 Fluc进行组成性共表达第一步(1),加入含有 LgBiT 蛋白的 ONE-GIo™ EX 试剂,定量 Fluc。其可裂解细胞,提供 Fluc 底物,并将 HiBi7标签转换为 NanoBiT®萤光素酶。第二步(2),向样品中加入NanoDLR® Stop&Glo®Reagent,淬灭 Fluc 信号并提供 NanoBiT® Substrate。

用 TK启动子驱动的 HiBiT-BRD4和 Fluc 的双顺反子表达构建体瞬时转染 HEK293 细胞。(A)用靶向 BRD4的PROTAC 化合物 dBET1 处理细胞,会导致 HiBiT-BRD4 特异性降解,而 Fluc对照不会发生降解。(B)另一方面用有毒化合物星形孢菌素处理,两种蛋白信号均出现了非特异性下降。


特点和优势


标签小(11 个氨基酸,1.3kDa)

●降低对融合蛋白伴侣功能的潜在影响

●大大简化了 CRISPR/Cas9 基因组敲入的工作流程


便于用 CRISPR/Cas9 进行内源性蛋白检测

●研究内源性调控下的蛋白质

●无需克隆

●方案可优化


简单、快速的检测方案

●一步均质检测(“加入-读数”)

●无需抗体,无洗涤步骤

●适用于高通量筛选


多种检测模式

●裂解终点法检测模式

●活细胞形式(细胞内和细胞外),具有可延长时程分析选项


灵敏度及定量

●Sub-attomole 级灵敏度,能够检测内源性水平的蛋白

●线性检测范围大(>7 logs)

产品列表


Product

Size

Cat. #

Nano-Glo®HiBiT 裂解检测系统

10 ml

N3030

100 ml

N3040

10x100 ml

N3050

Nano-Glo®HiBiT 细胞外检测系统

10 ml

N2420

100 ml

N2421

10x100 ml

N2422

Nano-Glo® HiBiT 印迹系统

100 ml

N2410

HiBiT 对照蛋白

100 µl

N3010


Product

Size

Cat. #

Nano-Glo® 活细胞测定系统

100 assays

N2011

1000 assays

N2012

10,000 assays

N2013

Nano-Glo® Vivazine™ 底物

0.1 ml

N2580

1 ml

N2581

10 ml

N2582

Nano-Glo® Endurazine™ 底物

0.1 ml

N2570

1 ml

N2571

10 ml

N2572

Nano-Glo® 扩展的活细胞底物试验包

0.2 ml

N2590

LgBiT 亚基的共表达

LgBiT Expression Vector [CMV / Hygro]

20 μg

N2681

LgBiT-LentiB3 Transfer Vector

20 μg

Please

Enquire

HaloTag®-LgBiT Expression Vector [CMV / Hygro]

20 μg

Please

Enquire

HEK293 LgBiT Cell Line (stable)

1 each

N2672

HeLa LgBiT Cell Line (stable)

2 vials

Please

Enquire

Jurkat LgBiT Cell Line (stable)

2 vials

Please

Enquire


Product

Size

Cat. #

HiBiT NanoDLR™

Nano-Glo® HiBiT Dual-Luciferase™ Reporter System

10ml

Please

Enquire

Nano-Glo® HiBiT Dual-Luciferase™ Reporter System

100ml

Please

Enquire

pBiT4.1-C [HiBiT-IRES-luc2/CMV/Blast] Vector

20 μg

Please

Enquire

pBit4.2-C [HiBiT-IRES-luc2/TK/Blastl Vector

20 μg

Please

Enquire

pBiT4.3-C [HiBiT-RES-luc2/PGK/Blast] Vector

20 μg

Please

Enquire

pBiT4.1-N [HiBiT-IRES-luc2/CMV/Blast] Vector

20 μg

Please

Enquire

pBit4.2-N [HiBiT-IRES-luc2/TK/Blastl Vector

20 μg

Please

Enquire

pBiT4.3-N [HiBiT-IRES-luc2/PGK/Blast] Vector

20 μg

Please

Enquire

HiBiT 融合载体

pBiT3.1-N [CMV/HiBiT/Blastl Vector

20 μg

N2361

pBiT3.1-C[CMV/HiBiT/Blastl Vector

20 μg

N2371

pBiT3.1-secN [CMV/HiBiT/Blastl Vector

20 μg

N2381

pFN38K HiBiT CMV-neo Flexi Vector

20 μg

N2401

pFC37K HiBiT CMV-neo Flexi° Vector

20 μg

N2391

pFN39K secHiBiT CMV-neo Flexi Vector

20 μg

N2411

自噬 LC3 HiBiT 报告基因检测系统

U2OS Autophagy LC3 HiBiT Reporter Cell Lineand Detection System

1 kit*

GA1050

HEK293 Autophagy LC3 HiBiT Reporter Cell Lineand Detection System

1 kit*

GA1040

Autophagy LC3 HiBiT Reporter Vector and Detection System

1 kit*

GA2550

Janelia Fluor 549 HaloTag Ligands

5ug

GA1110

3x5ug

GA1111

Janelia Fluor 646 HaloTag Ligands

5ug

GA1120

3x5ug

GA1121

* 包括 10mL Nano-Glo® HiBiT 裂解检测系统


Product

Size

HiBiT 靶细胞杀伤作用检测

LgBiT Protein

10ml

0.1ml

HaloTag"-HiBiT Vector [CAG / Blast]

2μg

HaloTag®-HiBiT Target Cells

Raji Cells, Propagation Model

2 vials

Ramos Cells, Propagation Model

2 vials

A549 Cells, Propagation Model

2 vials

H929 Cells, Propagation Model

2 vials

SK-BR-3 Cells, Propagation Model

1 vial

K562 Cells, Propagation Model

1 vial

U937 Cells, Propagation Model

1 vial

LDH-HiBiT Target Cells

Raji Cells, Propagation Model

2 vials

Raji CD19-KO Cells, Propagation Model

2 vials

Ramos Cells, Propagation Model

2 vials

Ramos CD19-KO Cells, Propagation Model

2 vials

OVCAR-3 Cells, Propagation Model

2 vials

SC-0V-3 Cells, Propagation Model

2 vials

A549 Cells, Propagation Model

2 vials

T2 Cells, Propagation Model

2 vials

PBMC ADCC Bioassays

PBMC ADCC Bioassay Kit (Raji)

1 kit*

PBMC ADCC Bioassay Kit (Ramos)

1 kit*

PBMC ADCC Bioassay Kit (A549)

1 kit*

PBMC ADCC Bioassay Kit (H929)

1 kit*

PBMC ADCC Bioassay Kit (SK-BR-3)

1 kit*

*包括细胞培养基、胎牛血清(FBS)和 10mL Nano-Glo® HiBiT 细胞外检测系统。


Product

Size

Cat. #

Anti-HiBiT Monoclonal Antibody

1x 100ug

N7200

5x 100ug

N7210

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