Anti-Neuropilin 1 antibody [EPR3113]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neuropilin 1 antibody [EPR3113] (AB81321)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling Neuropilin 1 with ab81321 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab81321 Anti-Neuropilin 1 antibody [EPR3113] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control. Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

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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) (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.

• ICC/IF

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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. 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. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IHC-P

Unknown

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. 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.

• Flow Cyt (Intra)

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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.

• ICC/IF

PubMed

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.

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

• IP

Lab

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) (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 (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

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Exposure time: 4s

• WB

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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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• WB

Lab

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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• WB

Lab

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] (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 ab269507 (knockout 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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 (<a href='/en-us/products/cell-lines/human-nrp1-knockout-a549-cell-line-ab269507'>ab269507</a>)

Lane 3:

MDA-MB-231 cell lysate at 20 µg

Lane 4:

SK-BR-3 cell lysate at 20 µg

Predicted band size: 103 kDa

Observed band size: 125-135 kDa

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• WB

Lab

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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• WB

Lab

Western blot - Anti-Neuropilin 1 antibody [EPR3113] (AB81321)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of rat heart lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab81321 (1/10,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique.

ab81321 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected under these conditions. This data was generated as part of a structured antibody stability study supporting Abcam’s transition from cold‑chain to ambient shipping.

All lanes:

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

All lanes:

Rat heart lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 100s

• WB

Lab

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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