Recombinant
RabMAb

Recombinant Anti-RAGE antibody [EPR21171] (ab216329)

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Overview

  • Product name

    Anti-RAGE antibody [EPR21171]
    See all RAGE primary antibodies

  • Description

    Rabbit monoclonal [EPR21171] to RAGE

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, WB, IHC-P, IHC-Fr, ICC/IF, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Recombinant fragment within Human RAGE aa 1-350. The exact sequence is proprietary.
    Database link: Q15109

  • Positive control

    • WB: Human fetal lung lysate; Mouse and rat lung lysates. IHC-P: Human, mouse and rat lung tissues. IHC-Fr: Mouse and rat lung tissues. ICC/IF: HEK-293T cells. Flow Cyt: HEK-293T cells. IP: Mouse lung lysate.

  • General notes

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab216329 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP 1/30.
WB 1/1000. Predicted molecular weight: 42 kDa.
IHC-P 1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IHC-Fr 1/500.

Perform heat mediated antigen retrieval by using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) before commencing with IHC staining protocol.
ICC/IF 1/100.
Flow Cyt 1/500.

Target

  • Function

    Mediates interactions of advanced glycosylation end products (AGE). These are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Acts as a mediator of both acute and chronic vascular inflammation in conditions such as atherosclerosis and in particular as a complication of diabetes. AGE/RAGE signaling plays an important role in regulating the production/expression of TNF-alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes. Interaction with S100A12 on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Interaction with S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53/TP53 signaling (By similarity). Receptor for amyloid beta peptide. Contributes to the translocation of amyloid-beta peptide (ABPP) across the cell membrane from the extracellular to the intracellular space in cortical neurons. ABPP-initiated RAGE signaling, especially stimulation of p38 mitogen-activated protein kinase (MAPK), has the capacity to drive a transport system delivering ABPP as a complex with RAGE to the intraneuronal space.

  • Tissue specificity

    Endothelial cells.

  • Sequence similarities

    Contains 2 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.

  • Cellular localization

    Secreted and Cell membrane.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Advanced glycosylation end product-specific receptor antibody
    • Ager antibody
    • DAMA 358M23.4 antibody
    • MGC2235 antibody
    • MGC22357 antibody
    • RAGE_HUMAN antibody
    • Receptor for advanced glycation end products antibody
    • Receptor for advanced glycosylation end products antibody
    see all

Images

  • Western blot - Anti-RAGE antibody [EPR21171] (ab216329)
    Western blot - Anti-RAGE antibody [EPR21171] (ab216329)
    All lanes : Anti-RAGE antibody [EPR21171] (ab216329) at 1/2000 dilution

    Lane 1 : Mouse lung lysates with 5% NFDM/TBST
    Lane 2 : Mouse brain lysates with 5% NFDM/TBST
    Lane 3 : Mouse kidney lysates with 5% NFDM/TBST
    Lane 4 : Mouse heart lysates with 5% NFDM/TBST
    Lane 5 : Mouse liver lysates with 5% NFDM/TBST
    Lane 6 : Mouse spleen lysates with 5% NFDM/TBST
    Lane 7 : Rat lung lysates with 5% NFDM/TBST
    Lane 8 : Rat brain lysates with 5% NFDM/TBST
    Lane 9 : Rat kidney lysates with 5% NFDM/TBST
    Lane 10 : Rat heart lysates with 5% NFDM/TBST
    Lane 11 : Rat spleen lysates with 5% NFDM/TBST

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 42 kDa
    Observed band size: 43 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    The expression profile and molecular mass observed is consistent with what has been described in the literature (PMID:16315007; PMID:18355449; PMID:18245812)

  • Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen rat lung tissue labeling RAGE with ab216329 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on alveolar epithelial cells, negative on the bronchial epithelial cells on rat lung tissue section is observed (PMID: 15173891).

    The nuclear counter stain is DAPI (blue).

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

  • Western blot - Anti-RAGE antibody [EPR21171] (ab216329)
    Western blot - Anti-RAGE antibody [EPR21171] (ab216329)
    All lanes : Anti-RAGE antibody [EPR21171] (ab216329) at 1/1000 dilution

    Lane 1 : Mouse lung lysate
    Lane 2 : Rat lung lysate
    Lane 3 : Human fetal lung lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
    Lane 3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

    Developed using the ECL technique.

    Predicted band size: 42 kDa
    Observed band size: 45,55 kDa why is the actual band size different from the predicted?



    Exposure times: Lane 1: 5 seconds; Lane 2: 10 seconds; Lane 3: 3 minutes.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

    The expression profile and molecular mass observed is consistent with what has been described in the literature (PMID:16315007; PMID:18355449; PMID:18245812). Full-length RAGE is not detected in rat and human lysates.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunohistochemical analysis of paraffin-embedded human lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on epithelial cells of human lung (PMID: 19592063; PMID: 26472810) is observed. Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly membranous staining on epithelial cells of mouse lung (PMID: 19592063; PMID: 26472810) is observed. Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly membranous staining on epithelial cells of rat lung (PMID: 19592063; PMID: 26472810) is observed. Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse lung tissue labeling RAGE with ab216329 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on alveolar epithelial cells, negative on the bronchial epithelial cells on mouse lung tissue section is observed (PMID: 15173891).

    The nuclear counter stain is DAPI (blue).

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

  • Immunocytochemistry/ Immunofluorescence - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunocytochemistry/ Immunofluorescence - Anti-RAGE antibody [EPR21171] (ab216329)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with Myc-tagged RAGE expression vector labeling RAGE with ab216329 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing positive staining in HEK-293T cells transfected with Myc-tagged RAGE expression vector. 

    The nuclear counter stain is DAPI (blue). Myc-Tag is detected with Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 647 Conjugate) (red) at 1/1000 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.

    Negative control: Myc-transfected HEK-293T cells.

  • Flow Cytometry - Anti-RAGE antibody [EPR21171] (ab216329)
    Flow Cytometry - Anti-RAGE antibody [EPR21171] (ab216329)

    Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with Myc-tagged RAGE expression vector labeling RAGE with ab216329 at 1/500 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

    Fresh cells were surface-stained with ab172730 and ab216329 respectively. Then fixed with 2% PFA for 15min and intracellular stained with anti-Myc tag antibody (Y axis). Only Myc+ population give positive signal.

  • Immunoprecipitation - Anti-RAGE antibody [EPR21171] (ab216329)
    Immunoprecipitation - Anti-RAGE antibody [EPR21171] (ab216329)

    RAGE was immunoprecipitated from 0.35 mg of mouse lung lysate with ab216329 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216329 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

    Lane 1: Mouse lung lysate 10 µg (Input). 
    Lane 2: ab216329 IP in mouse lung lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216329 in mouse lung lysate.

     

    Exposure time: 10 seconds.

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

References

This product has been referenced in:

  • Sun Z  et al. RAGE/galectin-3 yields intraplaque calcification transformation via sortilin. Acta Diabetol N/A:N/A (2019). Read more (PubMed: 30603868) »
  • Zhao Y  et al. High glucose-induced complement component 3 up-regulation via RAGE-p38MAPK-NF-?B signalling in astrocytes: In vivo and in vitro studies. J Cell Mol Med 22:6087-6098 (2018). Read more (PubMed: 30246940) »
See all 2 Publications for this product

Publishing research using ab216329? Please let us know so that we can cite the reference in this datasheet.

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