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使用敲除细胞株进行验证重组RabMAb

重组Anti-Robo1抗体[EPR23699-159] - BSA and Azide free (ab274385)

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Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Flow Cytometry - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Immunoprecipitation - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
  • Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23699-159] to Robo1 - BSA and Azide free
  • Suitable for: WB, Flow Cyt, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Unconjugated

概述

  • 产品名称

    Anti-Robo1抗体[EPR23699-159] - BSA and Azide free
    参阅全部 Robo1 一抗

  • 描述

    兔单克隆抗体[EPR23699-159] to Robo1 - BSA and Azide free

  • 宿主

    Rabbit

  • 经测试应用

    适用于: WB, Flow Cyt, IPmore details
    不适用于: ICC/IF,IHC-Fr or IHC-P

  • 种属反应性

    与反应: Mouse, Rat, Human

  • 免疫原

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • 阳性对照

    • WB: HeLa, HAP1 and HepG2 whole cell lysates; Mouse E14.5 brain and C57 E18 brain tissue lysates; Rat E14 brain tissue lysate; His-tagged human Robo1 recommbinant protein (aa660-897(Q883R)). IP: Mouse E14.5 brain tissue lysate. Flow cyt: HeLa cells.

  • 常规说明

    ab274385 is the carrier-free version of ab256791.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

性能

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab274385于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
WB
Use at an assay dependent concentration. Detects a band of approximately 120, 130, 250 kDa (predicted molecular weight: 181 kDa).
Flow Cyt
Use at an assay dependent concentration.
IP
Use at an assay dependent concentration.
说明
WB
Use at an assay dependent concentration. Detects a band of approximately 120, 130, 250 kDa (predicted molecular weight: 181 kDa).
Flow Cyt
Use at an assay dependent concentration.
IP
Use at an assay dependent concentration.
应用说明
Is unsuitable for ICC/IF,IHC-Fr or IHC-P.

靶标

  • 功能

    Receptor for SLIT1 and SLIT2 which are thought to act as molecular guidance cue in cellular migration, including axonal navigation at the ventral midline of the neural tube and projection of axons to different regions during neuronal development. In axon growth cones, the silencing of the attractive effect of NTN1 by SLIT2 may require the formation of a ROBO1-DCC complex. May be required for lung development.

  • 组织特异性

    Widely expressed, with exception of kidney.

  • 序列相似性

    Belongs to the immunoglobulin superfamily. ROBO family.
    Contains 3 fibronectin type-III domains.
    Contains 5 Ig-like C2-type (immunoglobulin-like) domains.

  • 翻译后修饰

    Ubiquitinated. May be deubiquitinated by USP33.

  • 细胞定位

    Membrane.
  • Information by UniProt

  • 数据库链接

    see all

  • 别名

    • Deleted in U twenty twenty antibody
    • DUTT 1 antibody
    • DUTT1 antibody
    • FLJ21882 antibody
    • H Robo 1 antibody
    • H-Robo-1 antibody
    • hRobo 1 antibody
    • Robo 1 antibody
    • Robo1 antibody
    • ROBO1_HUMAN antibody
    • Roundabout 1 antibody
    • Roundabout axon guidance receptor homolog 1 antibody
    • Roundabout homolog 1 antibody
    • Roundabout homolog1 precurser antibody
    • Roundabout1 antibody
    • SAX 3 antibody
    • SAX3 antibody
    see all

图片

  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

    Lane 1 : Wild-type U-87 MG cell lysate
    Lane 2 : ROBO1 knockout U-87 MG cell lysate
    Lane 3 : Wild-type HAP1 cell lysate
    Lane 4 : ROBO1 knockout HAP1 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 181 kDa
    Observed band size: 215 kDa why is the actual band size different from the predicted?



    Western blot: Anti-ROBO1 antibody [EPR23699-159] (ab256791) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab256791 was shown to bind specifically to ROBO1. A band was observed at 215 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in ROBO1 knockout cell line. The identity of the lower band is unknown. To generate this image, wild-type and ROBO1 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : ROBO1 CRISPR/Cas9 edited HAP1 cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : MCF7 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 181 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?



    This data was developed using the same antibody clone in a different buffer formulation (ab256791).

    Lanes 1 - 4: Merged signal (red and green). Green - ab256791 observed at 120 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

    ab256791 was shown to react with Robo1 in western blot. The band observed in the CRISPR/Cas9 edited lysate lane below 120 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab256791 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Flow Cytometry - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Flow Cytometry - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)

    This data was developed using ab256791, the same antibody clone in a different buffer formulation.

    Flow cytometric analysis of LoVo (Human colorectal adenocarcinoma epithelial cell) (Left) / HeLa (Human cervix adenocarcinoma epithelial cell) (Right) cells labelling Robo1 with ab256791 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

    Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

    Negative control: LoVo(PMID: 16740745).

    Gated on viable cells.

  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

    Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 2 : LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate
    Lane 3 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
    Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

    Predicted band size: 181 kDa
    Observed band size: 120-130 kDa why is the actual band size different from the predicted?



    This data was developed using ab256791, the same antibody clone in a different buffer formulation.

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The molecular weight observed is consistent with what has been described in the literature (PMID: 20471383, 16740745). The 120/130-kDa bands correspond to the Robo1 cleavage fragments from metalloprotease and/or gamma secretase activities (PMID: 20570941, 20471383).

    Negative control: LoVo (PMID: 16740745); Low expression: MCF7 (PMID: 16740745).

    Lysates used in this blot have experienced a freeze-thaw cycle.

    Exposure time: 10 seconds.

  • Immunoprecipitation - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Immunoprecipitation - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)

    This data was developed using ab256791, the same antibody clone in a different buffer formulation.

    Robo1 was immunoprecipitated from 0.35 mg Mouse E14.5 brain tissue lysate with ab256791 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256791 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.

    Lane 1: Mouse E14.5 brain tissue lysate 10 ug.

    Lane 2: ab256791 IP in Mouse E14.5 brain tissue lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256791 in mouse E14.5 brain tissue lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 1 second.

     

  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

    Lane 1 : Mouse E14.5 brain tissue lysate
    Lane 2 : C57 E18 brain tissue lysate
    Lane 3 : Rat E14 brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

    Predicted band size: 181 kDa
    Observed band size: 120,250 kDa why is the actual band size different from the predicted?



    This data was developed using ab256791, the same antibody clone in a different buffer formulation.

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The 120 kDa band corresponds to the Robo1 cleavage fragments from metalloprotease and/or gamma secretase activities (PMID: 20570941, 20471383).

    Lysates used in this blot have experienced a freeze-thaw cycle.

    Exposure time: 70 seconds.

  • Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Western blot - Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    All lanes : Anti-Robo1 antibody [EPR23699-159] (ab256791) at 1/1000 dilution

    Lane 1 : His-tagged human Robo1 recommbinant protein (aa660-897(Q883R))
    Lane 2 : His-tagged human Robo2 recommbinant protein (aa621-859)
    Lane 3 : His-tagged human Robo3 recommbinant protein (aa655-892)
    Lane 4 : His-tagged human Robo4 recommbinant protein (aa301-463)

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

    Predicted band size: 181 kDa
    Observed band size: 38 kDa why is the actual band size different from the predicted?



    This data was developed using ab256791, the same antibody clone in a different buffer formulation.

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    These recombinant proteins were made in house. The sample loaded onto lane 1 was purified Robo1 recombinant protein expressed from a mammalian - HEK-293 expression system. The samples loaded onto lanes 2-4 were E.coil extracts containing Robo2, Robo3 or Robo4 recombinant protein respectively.

    Exposure time: 26 seconds.

  • Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)
    Anti-Robo1 antibody [EPR23699-159] - BSA and Azide free (ab274385)

实验方案

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

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