Rab35 活性检测试剂盒类目 > 商铺首页 > 产品展示 > 活性检测试剂盒厂家_Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒
Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒 Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒

Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒

[Rab35kit

Rab35-GTP

active rab35

Rab35 assay

Rab35 kit

Rab35活性检测试剂盒

Rab35]

活性检测试剂盒厂家

价格: 电议

物流: 湖北 武汉市 洪山区| 卖家支付运费

可销售总量: 6000件

手机: 18064125815 邮箱: 2892088303@qq.com

传真: 027-87561033 地址: 湖北

在线询盘

提交询盘信息,一呼百应会为您匹配更多优质、合适的供应商!

*采购产品:

信息不能为空!请填写!

*采购量/单位:

采购量不能为空!

*期望价格:

价格不能为空!

*报价截止日期:

截止日期不能为空!

*联系人:

联系人不能为空!

*联系电话:

联系电话不能为空!

 

[Rab35kit Rab35-GTP active rab35 Rab35 assay Rab35 kit Rab35活性检测试剂盒 Rab35]

Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒
我想要了解:
请输入您要咨询的问题:
您的联系方式:

邮箱:

手机:

详细信息(Rab35 Activation Assay Kit Rab35 Assay Kit| Rab35 活性检测试剂盒)
  • 药准字:
  • 主要组成成分: Rab35
  • 性状: 液体
  • 适应症: 科研
  • 规格: 30μl
  • 有效期: 24
  • 批准文号:
  • 生产企业: NewEastBio
  • 货号: 82801
  • 免疫原: Mouse
  • 适用范围: 脊椎动物

Configuration-specific Monoclonal Antibody Based

Rab35 Activation Assay Kit

Catalog Number:82801

20 assays


Product Description


The Rab family of GTPases regulates eukaryotic vesicular membrane traffic. Rab35 is with both plasma

membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions.


Currently there is no direct assay to measure the activation of Rab35 GTPases.


NewEast Biosciences Rab35 Activation Assay Kit is based on the configuration-specific monoclonal

antibody that specifically recognizes Rab35-GTP, but not Rab Rab35-GDP. Given the high affinity of

monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This

assay provides the reliable results with consistent reproducibility.

These anti- Rab35-GTP monoclonal antibodies can also be used to monitor the activation of Rab35 in

cells and in tissues by immunohistochemistry.


NewEast Biosciences Rab35 Activation Assay Kit provides a simple and fast method to monitor the

activation of Rab35. Each kit provides sufficient quantities to perform 20 assays. 


Assay Principle


NewEast Biosciences Rab35 Activation Assay Kit bases on the configuration-specific anti-Rab35-GTP

monoclonal antibody to measure the active Rab35-GTP levels, either from cell extracts or from in vitro

GTPγS lo*g Rab35 activation assays. Briefly, anti-active Rab35 mouse monoclonal antibody will be

incubated with cell lysates containing Rab35-GTP. The bound active Rab35 will then be pulled down

by protein A/G agarose. The precipitated active Rab35 will be detected by immunoblot analysis using

anti- Rab35 rabbit polyclonal antibody. 


Kit Components


1. Anti-active Rab35, Mouse Monoclonal Antibody (Catalog No. 26922): One vial – 22 ?L (1

    mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

    specifically recognizes Rab35 -GTP from all vertebrates.

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

3. 5X Assay/Lysis Buffer (Catalog No. 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- Rab35, Rabbit Polyclonal Antibody (Catalog No. 21078): One vial – 100 ?L (1 mg/ml)

    in PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30303): One vial –100 ?l at 10 mM, use 5 ?L of GTPγS for

    GTP-labeling of 0.5 mL of cell lysate.

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

    GDP-labeling of 0.5 mL of cell lysate. 


Storage


Store all kit components at 4?C 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. 0.5 M EDTA, pH8.0

5. 1 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. PVDF or nitrocellulose membrane

11. Secondary Antibody

12. 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 ?g/mL leupeptin, and 10 ?g/mL aprotinin. 


Sample Preparation



Adherent Cells

1. Culture cells (one 10-cm plate, ~ 107cells) 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 27?-gauge syringe needle 3-4 times to shear the

    genomic DNA.

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

9. 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 ce*ifugation.

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 27?-gauge syringe needle 3-4 times to shear the

    genomic DNA.

8. Clear the lysates by ce*ifugation 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 Lo*g for positive and negative co*ols

    Note: In vivo stimulation of cells will activate approximately 10% of the *ailable Rab35, whereas

    in vitro GTPγS protein lo*g will activate nearly 90% of the Rab35.

1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 ?g of purified Rab35 protein).  

2. To each tube, add 20 ?l of 0.5 M EDTA (to 20 mM final conce*ation).

3. Add 5 ?l of 100 X GTPγS (to 100 ?M, final conce*ation) to one tube (positive co*ol).

4. Add 5 ?l of 100 X GDP (to 1 mM, final conce*ation) to the second tube (negative co*ol).

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

6. Stop lo*g by placing the tubes on ice and adding 32.5 ?l of 1 M MgCl2 (to 60 mM, final

    conce*ation). 


Assay Procedure 


I. Active Rab35 Pull-Down Assay

1. Aliquot 0.5 – 1 mL of cell lysate to a microce*ifuge tube.

2. Adjust the volume of each sample to 1 mL with 1X Assay/Lysis Buffer.

3. Add 1 ?l anti-active Rab35 monoclonal antibody to the tube.

4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.

5. Quickly add 20 ?L of resuspended bead slurry to each tube.

6. Incubate the tubes at 4 °C for 1 hour with gentle agitation.

7. Pellet the beads by ce*ifugation for 1 min at 5,000 x g.

8. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.

9. Wash the bead 3 times with 0.5 mL of 1X Assay/Lysis Buffer, ce*ifuging and aspirating
         each time.

10. After the last wash, pellet the beads and carefully remove all the supernatant.

11. Resuspend the bead pellet in 20 ?L of 2X reducing SDS-PAGE sample buffer.

12. Boil each sample for 5 minutes.

13. Ce*ifuge each sample for 10 seconds at 5,000 x g.


II. Electrophoresis and Transfer

1. Load 15 ?L/well of pull-down supernatant to a polyacrylamide gel (17%). Also, it’s

    recommended to include a pre-stained MW standard (as an indicator of a successful transfer in

    step 3).

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. 


III. Immunoblotting and Detection (all steps are at room temperature, with agitation)

1. Following the electroblotting step, immerse the PVDF membrane in * Methanol for 15

    seconds, and then allow it to dry at room temperature for 5 minutes.

    Note: If Nitrocellulose is used instead of PVDF, this step should be skipped.

2. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature

    with constant agitation.

    Incubate the membrane with anti- Rab35 polyclonal antibody, freshly diluted 1:50~1000

    (depending on the amount of Rab35 proteins in your samples) in 5% non-fat dry milk or 3%

    BSA/TBST, for 1-2 hr at room temperature with constant agitation or at 4oC overnight.

3. Wash the blotted membrane three times with TBST, 5 minutes each time.

4. Incubate the membrane with a secondary antibody (e.g. Goat Anti-Rabbit IgG, HRP-conjugate),

    freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA/TBST, for 1 hr at room temperature

    with constant agitation.

5. Wash the blotted membrane three times with TBST, 5 minutes each time.

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


Example of Results


The following figure demonstrates typical results seen with NewEast Biosciences Rab35 Activation

Assay Kit. One should use the data below for reference only. 

QQ截图20191118145815.png

Rab35 activation assay. Purified GST-tagged Rab35 proteins (Cat. #10132) were immunoprecipitated

with the anti-active Rab35 monoclonal antibody (Cat. #26922) after treated with GDP (lane 1) or GTPγS

(lane 2), and was blotted with anti-Rab35 polyclonal antibody(Cat. #21078). Input co*ol is shown in

the bottom panel. 


Related Products
Catalog# Name Size Price
82801 Rab35 Activation Assay Kit  20 assays  ¥6800  
26922 Active Rab35-GTP Monoclonal Antibody  30 μL  ¥4800   
21078 Anti-Rab35 Rabbit Polyclonal Antibody  100 μL  ¥2600  



Publications:
1.  VASP promotes TGF-β activation of hepatic stellate cells by regulating Rab11 dependent plasma membrane targeting of TGF-β receptors
    Hepatology Volume 61, Issue 1, pages 361–374, January 2015
2.  Rab5 Activity Regulates GLUT4 Sorting into Insulin-Responsive and Non-Insulin-Responsive Endosomal Compartments: A Potential Mechanism for Development of Insulin Resistance
    Endocrinology. 2014 Sep;155(9):3315-28
标签: Rab3... Rab3... acti... Rab3... Rab3...   Rab35kit Rab35-GTP active rab35 Rab35 assay Rab35 kit Rab35活性检测试剂盒 Rab35   活性检测试剂盒   活性检测试剂盒厂家
企业诚信档案
武汉纽斯特生物技术有限公司
1
VIP会员 营业执照 法人身份 手机认证 证书2张 商铺1年

注册资金:尚未完善

联系人:乐经理

固话:027-87561033

移动手机:18064125815

企业地址:湖北 洪山区

最新产品
Anti BRaf(V600E) Mouse Monoclonal AntibodyBRaf(V600E)点突变抗体图片

Anti BRaf(V600E)...

电议
Sar1 Activation Assay Kit Sar1 kit活性检测试剂盒图片

Sar1 Activation ...

电议
RheB Activation Assay Kit RheB kit活性试剂盒图片

RheB Activation ...

电议
Ras Activation Assay Kit Ras kit活性检测试剂盒图片

Ras Activation A...

电议
RalA Activation Assay Kit |RalA Assay Kit图片

RalA Activation ...

电议
友情链接
推荐采购信息
您好,欢迎来到一呼百应! 
登录 |免费注册
搜本站
武汉纽斯特生物技术有限公司
1 |
一呼百应推广
扫码访问移动版商铺
产品分类
首 页 产品展示 铺主推荐 公司新闻 公司简介 联系我们

快捷导航

在线询盘
在线QQ
电话联系
乐经理 18064125815 固话:尚未完善
返回顶部
古代验下面怎么验的 结婚后天天晚上搞到半夜,国产盗摄精品