Mitofusin1 Rabbit Recombinant mAb

Filter:

  • WB
规格 价格 库存 购买数量
20ul 447.49 现货
100ul 1500.19 现货
更大包装 有超大折扣 点击询价
 

400-668-6834

info@selleck.cn

 

使用信息

抗体应用 WB IP FC
稀释比例
WB IP FCM
1:1000~1:5000 1:20 1:20
反应性 Human
MW (kDa) 84kDa
抗体类型 Rabbit
浓度 1mg/ml
储存液配方 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
储存条件
(自收到货起)
Store at –20°C.

Datasheet & SDS

生物描述

特异性

Mitofusin1 Rabbit Recombinant mAb可检测内源性的总Mitofusin1水平。

背景

线粒体融合蛋白是一种线粒体跨膜GTP酶,其功能是调节线粒体融合,这一过程发生在线粒体分裂的同时,是维持线粒体结构和遗传完整性所必需的。在哺乳动物中已经发现了两种线粒体融合蛋白,mitofusin-1和mitofusin-2,它们具有60%的氨基酸同源性,并协同作用调节线粒体融合。

实验方法

WB

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

IP

IP

Sample preparation

1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
2. Remove PBS and add ice-cold 1X cell lysis buffer to each plate and incubate on the ice for 5 min.
3. Scrape cells off the plate and transfer to themicrocentrifuge tubes. Keep on the ice.
4. Sonicate on the ice three times for 5 sec each.
5. Centrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. If necessary, store itin -80°C.

Cell Lysate Pre-Clearing (Optional)

Pre-clearing the lysate can help reduce non-specific binding and reduce background.

1. Vortex to mix beads.
2. Add 10–30 µl of 50% Protein Aagarose bead slurry to 200 µl cell lysate at 1 mg/ml.
3. Incubate with rotation at 4°C for 30–60 min.
4. Centrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
5. Proceed to the next part.

Immunoprecipitation

1. Add primary antibody at the appropriate dilutionto cell lysat. Incubate with gentle agitation or rotation overnight at 4°C.
2. Add protein Aagarose(10–30 µl of 50% bead slurry). Incubate with gentle agitation or rotation for 1–3 hr at 4°C.
3. Centrifuge for 30 sec at 4°C and discard the supernatant. Wash the pellet five times with 500 µl of 1X cell lysis buffer. Keep on the ice.
4. Continue with sample analysis.

Sample Analysis

a. Western Immunoblotting

1. Resuspend the pellet with SDS sample buffer. Vortex, then centrifuge for 30 sec at 14,000 x g.
2. Heat the samples to 95–100°C for 2-5 min and centrifuge for 1 min at 14,000 x g.
3. Load the appropriate amount ofsamples on a 4–20% gel for SDS-PAGE.
4. Analyze sample by western blot.

b. Kinase Assay

1. Wash the pellet twice with 500 µl 1X kinase buffer. Keep on the ice.
2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate the reaction by adding SDS sample buffer. Vortex to mix, then centrifuge for 30 sec.
5. Transfer supernatantcontaining phosphorylated substrate to another tube.
6. Heat the sample to 95–100°C for 2–5 min and centrifuge for 1 min at 14,000 x g.Load the sample on SDS-PAGE.

技术支持

在订购、运输、储存和使用我们的产品的任何阶段,您遇到的任何问题,均可以通过拨打我们的热线电话400-668-6834,或者技术支持邮箱tech@selleck.cn,直接联系到我们。我们会在24小时内尽快联系您。

* 必填项

请输入您的姓名
请输入您的邮箱地址 请输入一个有效的邮箱地址
请写点东西给我们
在线咨询
联系我们