Ras 活性检测试剂盒

厂商 ：武汉费斯德生物科技有限公司

湖北 武汉市
  • 主营产品：
  • G蛋白活性抗体
  • 点突变抗体
  • 非乙酰化CAMP和C
联系电话 ：18162422833
商品详细描述

品牌: 美国 NewEast Biosciences
货号: 81101
规格: 30 Assays
产品全名: Ras Pull-Down Pull-Down Assay Kit
应用: Pull-Down
监测: Active Ras-GTP levels
、Ras Pull-Down Activation Assay Kit

Cat. # 81101

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. Ras belongs to the Ras sub-family of GTPases that regulate cell growth, cell motility, and gene tranion. GTP binding increases the activity of Ras, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Ras proteins is assayed with the binding of GTP-bound Ras to the Ras-binding domain (RBD) of Raf protein kinase. This method is based on the observation that the active, GTP-bound Ras could bind to the RBD of Raf. However, the reproducibility of this method is poor. This is partially due to the relatively quick hydrolysis of GTP to GDP during the assay procedure, and the low binding affinity of RBD to Ras-GTP.
The Ras Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Ras-GTP, but not Ras-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a much shorter time. This assay provides the reliable results with consistent reproducibility.
B. Assay Principle
The Ras Activation Assay Kit uses configuration-specific anti-Ras-GTP Mouse monoclonal antibody to measure Ras-GTP levels in cell extracts or in vitro GTPγS loading Ras activation assays. Anti-Ras-GTP mouse monoclonal antibody is first incubated with cell lysates containing Ras-GTP. Next, the GTP-bound Ras is pulled down by protein A/G agarose. Finally, the precipitated Ras-GTP is detected through immunoblot analysis using Anti-Ras Rabbit Polyclonal Antibody.
The anti-Ras-GTP monoclonal antibody can also be used to monitor the activation of Ras in cells and in tissues by immunohistochemistry.
C. Kit Components
1. Anti-Ras-GTP Mouse Monoclonal Antibody (Cat. # 26909): One vial – 35 ?L (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Ras-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): One vial – 600 ?L of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Ras Rabbit Polyclonal Antibody (Cat. # 21021): One vial – 50 ?L (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): One vial – 50 ?l at 10 mM, use 5 ?L of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): One vial – 50 ?l at 100 mM, use 5 ?L of GDP for GDP-labeling of 0.5 mL of cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 ?L (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
D. Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4°C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE sample buffer
7. Electrophoresis and immunoblotting systems
8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%  Tween-20)
9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Ras Activation Assay Kit. For reference only.

Ras Activation Assay Kit


Ras Activation Assay. Purified Ras protein was loaded with GDP (lane 1) or GTPγS (lane 2). These proteins were incubated with an anti-Ras-GTP monoclonal antibody (Cat. # 26905) (top panel). The precipitated active Ras was immunoblotted with an anti-Ras rabbit polyclonal antibody (Cat. # 21021). The bottom panel shows the Western blot with anti-Ras of the used Ras-GDP and Ras-GTPγS.

Assay Procedure

A. Reagent Preparation
1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 ?g/mL leupeptin, or 10 ?g/mL aprotinin.
B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the 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 (See Reagent Preparation) 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 viscous and difficult to pipette. If this occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.
Adherent Cells
1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count and then pellet the cells through 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 (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.
C. In vitro GTPγS/GDP Protein for Positive and Negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Ras, whereas in vitro GTPγS protein loading will activate nearly 90% of Ras.
1. Aliquot 0.5 mL of cell extract (or 1 ?g of purified Ras protein) into two microcentrifuge tubes.
2. To each tube, add 20 ?L of 0.5 M EDTA (final concentration of 20 mM).
3. Positive control: add 5 ?L of 100 X GTPγS (Cat. # 30302) to the 1st tube
4. Negative control: add 5 ?L of 100 X GDP (Cat. # 30304) to the 2nd tube.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 ?L of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 ?L anti-Ras-GTP antibody (Cat. # 26909).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 ?L of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet.
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 ?L of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 ?L/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.
5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with Anti-Ras Rabbit Polyclonal Antibody (Cat. # 21021), which has been freshly diluted 1: 50~500 (depending on the amount of Ras proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.

11. Use the detection method of your choice such as ECL.


武汉费斯德生物科技有限公司是美国NewEast Biosciences在中国的办事处。NewEast Biosciences 在十二年前率先研发俩种独特的抗体。这俩种抗体仅仅识别活性的GTP酶或者突变的Oncogene。 GTP酶涉及(1)响应细胞表面受体激活的信号转导，包括跨膜受体，例如介导味觉、嗅觉和视觉的那些，（2）核糖体的蛋白质生物合成，（3）调节细胞分化、增殖、分裂和运动，（4）蛋白质通过膜的易位，（5）细胞内囊泡的运输，以及囊泡介导的分泌和摄取，通过GTP酶控制囊泡外壳组装。Oncogene侧是诱发癌症的基因。

我公司将向你提供以下三种抗体或者试剂盒: (1) 仅识别 GTP酶的活性构型的产品, 它可以让你能够量化GTP酶在细胞中的活性和分布。(2) 识别突变 Oncogene蛋白, 但不认识相应野生型的抗体。 (3) 对 cAMP 和 cGMP 具有超亲和力（无需乙酰化）ELISA检测试剂盒。这些产品被将近一千篇同行评议的文章所引用。

2021年武汉费斯德生物科技有限公司和NewEast Biosciences收购了武汉纽斯特生物技术有限公司所有的产品和细胞株。因此, 纽斯特不再拥有这些产品销售权。如果您在 2022 年 6 月 1 日之后从 纽斯特购买相同的产品, 你不应该在你的论文中引用 纽斯特的产品为NewEast Biosciences产品。

 ** 本公司销售的所有产品仅供实验科研使用，不用于人体及临床诊断。


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