Anti-Neuropilin 1 antibody [EPR3113]

11 product images

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  Image from Pérez-Lozano ML et al., PLoS One. 2013;8(4):e60776. Fig 5.; doi: 10.1371/journal.pone.0060776.

  Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  The expression of Neuropilin 1, VEGFR-2, and VEGF was analyzed by immunofluorescence microscopy in omentum and effluent-derived mesothelial cells (MCs). MCs were double stained for Neuropilin 1 (green) and VEGFR-2 (red), and single stained for VEGF (green). Nuclei were stained with DAPI. Neuropilin 1 and VEGF show a membrane distribution in omentum and epithelioid MCs (b, c,h, i). During in vitro (e, f) and ex vivo (k, l) MMT both proteins change their localization and are internalized. The expression of VEGFR-2 is down-regulated but it does not show differences in localization during in vitro (a, d) and ex vivo (g, j) MMT.

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  Immunoprecipitation - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Neuropilin 1 was immunoprecipitated from 0.35mg mouse heart lysate with ab81321 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab81321 at 1/1000 dilution (0.77 μg/mL). VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary antibody at 1/1000 dilution.
  

  Lane 1: Mouse heart tissue lysate 10 μg
  Lane 2: Mouse heart tissue lysate
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab81321 in mouse heart lysate.

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

  All lanes: Immunoprecipitation - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Predicted band size: 103 kDa

  Observed band size: 120 kDa

  Exposure time: 4s

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  Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  False colour image of Western blot: Anti-Neuropilin 1 antibody [EPR3113] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab81321 was shown to bind specifically to Neuropilin 1. A band was observed at 125/135 kDa in wild-type A549 cell lysates with no signal observed at this size in NRP1 knockout cell line Human NRP1 knockout A549 cell line ab269507 (knockout cell lysate Human NRP1 knockout A549 cell lysate ab269669). To generate this image, wild-type and NRP1 knockout A549 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 (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: NRP1 knockout A549 cell lysate at 20 µg

  Lane 2: Western blot - Human NRP1 knockout A549 cell line (Human NRP1 knockout A549 cell line ab269507)

  Lane 3: MDA-MB-231 cell lysate at 20 µg

  Lane 4: SK-BR-3 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 103 kDa

  Observed band size: 125-135 kDa

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  Flow Cytometry (Intracellular) - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Intracellular Flow Cytometry analysis of MCF7 cells labelling Neuropilin 1 with purified ab81321 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

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  Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Blocking and dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/10000 dilution

  Lane 1: Mouse heart tissue lysate at 20 µg

  Lane 2: Rat heart tissue lysate at 20 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

  Predicted band size: 103 kDa

  Observed band size: 120 kDa

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  Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Blocking and dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/10000 dilution

  All lanes: Human heart tissue lysate at 20 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

  Predicted band size: 103 kDa

  Observed band size: 120 kDa

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  Flow Cytometry (Intracellular) - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Overlay histogram showing HepG2 cells stained with unpurified ab81321 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab81321, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HepG2 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Neuropilin 1 with purified ab81321 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Blocking and dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/2000 dilution

  All lanes: Human placenta tissue lysate at 20 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

  Predicted band size: 103 kDa

  Observed band size: 120 kDa

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  Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  All lanes: Western blot - Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/1000 dilution

  Lane 1: Human placenta lysate at 10 µg

  Lane 2: HUVEC cell lysate at 10 µg

  Lane 3: HepG2 cell lysate at 10 µg

  Lane 4: Mouse heart tissue lysate at 10 µg

  Lane 5: Mouse kidney tissue lysate at 10 µg

  Lane 6: Rat heart tissue lysate at 10 µg

  Lane 7: Rat kidney tissue lysate at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

  Predicted band size: 103 kDa

  Observed band size: 120 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Neuropilin 1 antibody [EPR3113] (ab81321)

  Immunocytochemistry/Immunofluorescence analysis of HUVEC cells labelling Neuropilin 1 with purified ab81321 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

  Control 1: primary antibody (1/250) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).