Gαi Assay Kit--性能参数，报价/价格，图片

基本信息

产品名称：

Gαi Assay Kit

英文名称：

Gαi Activation Assay Kit

国产/进口：

进口

产地/品牌：

USA/NeweastBio

型号：

80301

参考报价：

销售商：

武汉纽斯特生物技术有限公司

总点击数：

1165

更新日期：

2021-12-29

产品类别：

其他生物试剂

性能参数

Configuration-specific Monoclonal Antibody Based

Gαi Activation Assay Kit

Catalog Number： 80301

20 assays

Product Description

A structurally diverse repertoire of ligands, from photons to large peptides, activates G protein-coupled

receptors (GPCRs) to elicit their physiological functions. Ligand-bound GPCRs, in turn, function as

guanine nucleotide exchange factors catalyzing the exchange of GDP bound on the Gα subunit with

GTP in the presence of Gβγ, causing the dissociation of the Gα subunit from the Gβγ dimer to form

two functional units (Gα and Gβγ). Both Gα and Gβγ subunits signal to various cellular signaling

pathways. Based on the sequence and functional homologies, G proteins are grouped into four families:

Gs, Gi, Gq, and G12.

Gαi family is the largest family of G proteins. They relay signals from many GPCRs to regualte

various biological functions. There were no direct methods to measure the activation of Gαi proteins

by receptors (until this assay kit). Most reports used one of the downstream pathway, i.e. the

inhibition of adenylyl cyclases, as a readout. Alternatively, sensitivity to pertussis toxin (PTX) was

used as an indicator of possible Gαi proteins invovled in a signaling pathway.

NewEast Biosciences Gαi Activation Assay Kit provides a direct measurement of the activation of Gαi

proteins. This is a simple and fast tool to monitor the activation of Gαi. Each kit provides sufficient

quantities to perform 20 assays.

NewEast Biosciences Gαi Activation Assay Kit is based on the monoclonal antibody specifically

recognizing the active GTP-bound Gαi proteins. This monoclonal antibody has much lower affinity

towards the inactive Gαi proteins. Therefore, after activation by receptor signals, active GTP-bound

Gαi proteins could be immunoprecipitated by this monoclonal antibody and further quantified by

western blot with another anti- Gαi antibody.

Assay Principle

NewEast Biosciences Gαi Activation Assay Kit is an immunoprecipitation/western blot assay to

measure the levels of active GTP-bound Gαi proteins, either from cell extracts or from in vitro GTPγS

loaded Gαi proteins. Briefly, the anti-active Gαi monoclonal antibody will specifically bind to active

Gαi protein. This antibody/ Gαi complex will then be pulled down by protein A/G agarose. The

precipitated active Gαi proteins will be detected by immunoblots with another anti-Gαi antibody.

Kit Components

1. Anti-active Gαi, Mouse Monoclonal Antibody (Catalog No. 26901): One vial – 22 μL (1

mg/mL) in PBS, pH 7.4, contained 50% glycerol. This antibody specifically recognizes

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 μL of 50% slurry.

3. 5X Assay/Lysis Buffer (Catalog No. 30303): One bottle – 30 mL of 250 mM Tris-HCl, pH 7.4,

750 mM NaCl, 5 mM EDTA, 5% Triton X-100.

4. Anti-Gαi, Mouse Monoclonal Antibody (Catalog No. 26003): One vial – 22 μL(1 mg/mL) in

PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): One vial –100 μL at 10 mM, use 5 μL of GTPγS for

GTP-labeling of 0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 μL at 100 mM, use 5 μL of GDP for

GDP-labeling of 0.5 mL of cell lysate.

Storage

Store all kit components at 4ºC until their expiration dates.

Materials Needed but Not Supplied

1. Stimulated and non-stimulated cell lysates

2. Protease inhibitors

3. 4 °C tube rocker or shaker

4. 1 M MgCl2

5. 2X reducing SDS-PAGE sample buffer

6. Electrophoresis and immunoblotting systems

7. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05 %

Tween-20)

8. Immunoblotting blocking buffer (TBST containing 5 % Non-fat Dry Milk or 3 % BSA)

9. PVDF or nitrocellulose membrane

10. Secondary Antibody

11. ECL Detection Reagents

Reagent Preparation

• 1X Assay/Lysis Buffer: Mix the 5X Stock briefly and dilute to 1X in deionized water. Just prior to

usage, add protease inhibitors such as 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin.

Sample Preparation

Adherent Cells

1. Culture cells (one 10-cm plate, ~ 107 cells) to approximately 80-90 % confluence. Stimulate

cells with activator or inhibitor as desired.

2. Aspirate the culture media and wash twice with ice-cold PBS.

3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cells (0.5

- 1 mL per 10 cm tissue culture plate).

4. Place the culture plates on ice for 10-20 minutes.

5. Detach the cells from the plates by scraping with a cell scraper.

6. Transfer the lysates to appropriate size tubes and place on ice.

7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this

occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the

genomic DNA.

Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

8. Collect the supernatant and store samples (~1-2 mg of total proteins) on ice for immediate use,

or snap freeze and store at - 70 °C for future use.

Suspension Cells

1. Culture cells and stimulate with activator or inhibitor as desired.

2. Perform a cell count, and then pellet the cells by centrifugation.

3. Aspirate the culture media and wash twice with ice-cold PBS.

4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cell

pellet (0.5 – 1 mL per 1 x 107cells).

5. Lyse the cells by repeated pipetting.

6. Transfer the lysates to appropriate size tubes and place on ice.

7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this

occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the

genomic DNA.

8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

9. Collect the supernatant and store samples on ice for immediate use, or snap freeze and store at -

70 °C for future use.

In vitro GTPγS/GDP Protein Loading for positive and negative controls

Note: In vivo stimulation of cells with receptor ligands might activate ~10 % of the available Gαi

proteins, whereas in vitro GTPγS loading could activate ~50 % of the Gαi proteins that can be

activated.

1. Aliquot 0.5 mL of each cell extract to two microfuge tubes.

2. To each tube, add 5 μL of 1M MgCl2 (to 10 mM final concentration).

3. Add 5 μL of 100X GTPγS (to 100 μM, final concentration) to one tube (positive control).

4. Add 5 μL of 100X GDP (to 1 mM, final concentration) to the second tube (negative control).

5. Incubate the tubes at 30°C for 90 minutes with agitation.

公司简介

武汉纽斯特生物技术有限公司是一家集生物医药产品的研发、生产、销售及技术服务为一体的中外合资企业，公司位于武汉东湖高新技术开发区光谷生物城创新基地, 研发实验室占地面积1500平方米。公司研发团队30余人，其中博士4人，硕士10余人。公司自2006年创办以来，一直致力于生物医药领域高技术含量的产品的研发和销售，并始终站在生物医药领域的最前沿，以细胞信号转导相关的创新药物筛选试剂盒, 癌症诊断试剂盒, 临床前评价药物对重要靶器官毒性的生物标志物的检测试剂盒以及个性化指导用药检测试剂盒为主要研发方向。公司现已建立起稳定完善的研发技术平台，研发出几十种具有国际独创性的，拥有完全自主知识产权的新药筛选试剂盒和癌症诊断试剂盒，并且迅速占领销售国际市场。公司已建立起活性蛋白质表达和纯化技术平台、结构特异性单克隆抗体和多克隆抗体研发技术平台、癌症分子诊断试剂盒研发技术平台等，具备完善的生产研发和质量控制体系，同时也初步建立起国内外销售网络，并与国内外许多大型的知名制药企业以及各重点高校科研院所建立了业务联系，逐步建立并形成了产——研——销相结合良性可持续发展的新型经济模式。

公司目前的经营范围和在研合作项目有：多克隆抗体，单克隆抗体，多肽合成定制服务,cGMP和cAMP单克隆抗体，ELISA试剂盒,癌症早期诊断试剂盒,癌症突变位点检测试剂盒,G 蛋白活性检测试剂盒,各类细胞因子检测试剂盒,小分子检测试剂盒。

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