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使用敲除细胞株进行验证重组RabMAb

重组Anti-Rab5抗体[EPR21801] - Early Endosome Marker (ab218624)

Submit a review Submit a question References (14)
Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Immunoprecipitation - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
  • Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR21801] to Rab5 - Early Endosome Marker
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 568 Alexa Fluor® 594 Alexa Fluor® 647 Alkaline Phosphatase APC Carrier Free HRP PE

概述

  • 产品名称

    Anti-Rab5抗体[EPR21801] - Early Endosome Marker
    参阅全部 Rab5 一抗

  • 描述

    兔单克隆抗体[EPR21801] to Rab5 - Early Endosome Marker

  • 宿主

    Rabbit

  • 特异性

    This antibody could cross-react with RAB5C, but affinity is very low.

  • 经测试应用

    适用于: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IPmore details

  • 种属反应性

    与反应: Mouse, Rat, Human

  • 免疫原

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 阳性对照

    • WB: HAP1, HeLa, MCF7 and Neuro-2a whole cell lysates; Human fetal brain, fetal heart and fetal spleen lysates; Rat and mouse brain and heart lysates. IHC-P: Human kidney and cerebrum tissues; Mouse kidney tissue; Rat liver tissue. ICC/IF: NIH/3T3, HeLa and HAP1 cells. Flow Cyt (intra): MCF7 cells, Hap1 cells. IP: MCF7 whole cell lysate.

  • 常规说明

    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ab218624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
Flow Cyt (Intra)
1/50.
WB
1/1000. Detects a band of approximately 25 kDa (predicted molecular weight: 24 kDa).
IHC-P
1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF
1/1000.
IP
1/30.
说明
Flow Cyt (Intra)
1/50.
WB
1/1000. Detects a band of approximately 25 kDa (predicted molecular weight: 24 kDa).
IHC-P
1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF
1/1000.
IP
1/30.

靶标

图片

  • Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Flow cytometry overlay histogram showing wild-type Hap1 (green line) and Rab5A knockout Hap1 stained with ab218624 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab218624) (1x 106 in 100μl at 0.2 μg/ml (1/10700)) for 30min at 22°C.

    The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

    Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, Rab5A knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

    Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

    This antibody gave a positive signal in Hap1 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

     

  • Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    All lanes : Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624) at 1/1000 dilution

    Lane 1 : Wild type HAP1 whole cell lysate
    Lane 2 : Rab5 knockout HAP1 whole cell lysate
    Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 24 kDa
    Observed band size: 25 kDa why is the actual band size different from the predicted?


    Exposure time: 15 seconds


    ab218624 was shown to specifically react with Rab5 in wild-type HAP1 cells as signal was lost in Rab5 knockout cells. Wild-type and Rab5 knockout samples were subjected to SDS-PAGE. Ab218624 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrumentusing the ECL technique.

  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed wild-type and RAB5A KO HAP1 cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing no staining in RAB5A KO HAP1 cell line and granular cytoplasmic staining in parental HAP1 cell line.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Western blot - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    All lanes : Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624) at 1/1000 dilution

    Lane 1 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate
    Lane 2 : Human fetal brain lysate
    Lane 3 : Human fetal heart lysate
    Lane 4 : Human fetal spleen lysate
    Lane 5 : Rat brain lysate
    Lane 6 : Rat heart lysate
    Lane 7 : Mouse brain lysate
    Lane 8 : Mouse heart lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 24 kDa
    Observed band size: 25 kDa why is the actual band size different from the predicted?



     

    Exposure time :  Lane 1: 5 seconds; Lanes 2/8: 15 seconds; Lanes 3/4: 6 seconds; Lanes 5/7: 3 seconds;  Lane 6: 26 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human cerebrum is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in mouse kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in rat liver is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in NIH/3T3 cell line.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

     

  • Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunocytochemistry/ Immunofluorescence - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed  HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in HeLa cell line.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Flow Cytometry (Intracellular) - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (human breast adenocarcinoma cell line) cell line labelling Rab5 with ab218624 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled 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. 

     

     

  • Immunoprecipitation - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Immunoprecipitation - Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

    Rab5 was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma cell line) whole cell lysate with ab218624 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab218624 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

    Lane 1: MCF7 whole cell lysate 10 μg (input).

    Lane 2: ab218624 IP in MCF7 whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab218624 in MCF7 whole cell lysate.

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

    Exposure time: 1 second.

     

  • Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)
    Anti-Rab5 antibody [EPR21801] - Early Endosome Marker (ab218624)

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

文献 (14)

发表研究结果有使用 ab218624？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab218624 被引用在 14 文献中.

  • de Leeuw SM  et al. APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes. Stem Cell Reports 17:110-126 (2022). PubMed: 34919811
  • Scaramuzzino C  et al. Calcineurin and huntingtin form a calcium-sensing machinery that directs neurotrophic signals to the nucleus. Sci Adv 8:eabj8812 (2022). PubMed: 34985962
  • Barreto BR  et al. Cocaine Modulates the Neuronal Endosomal System and Extracellular Vesicles in a Sex-Dependent Manner. Neurochem Res 47:2263-2277 (2022). PubMed: 35501523
  • Lee JH  et al. Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci 25:688-701 (2022). PubMed: 35654956
  • Rovelet-Lecrux A  et al. Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease. Acta Neuropathol Commun 9:196 (2021). PubMed: 34922638
View all Publications for this product

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