Recombinant
RabMAb

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

Overview

  • Product name
    Anti-Neuropilin 1 antibody [EPR3113]
    See all Neuropilin 1 primary antibodies
  • Description
    Rabbit monoclonal [EPR3113] to Neuropilin 1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, IP, Flow Cyt, IHC-Fr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Monkey, Common marmoset
  • Immunogen

    Synthetic peptide within Human Neuropilin 1 aa 900 to the C-terminus (intracellular). The exact sequence is proprietary.
    Database link: O14786
    (Peptide available as ab189308)

  • Positive control
    • WB: HUVEC and HepG2 whole cell lysate (ab7900), human placenta, kidney and heart, mouse heart and kidney and rat heart and kidney tissue lysates. IHC-P: Human liver tissue; Human peritoneal specimens; Rat brain tissue; Mouse brain tissue. ICC/IF: MCF7 and HUVEC cells; Omentum and effluent-derived mesothelial cells; COS1 fibroblast-like cell line derived from monkey kidney tissue . Flow Cyt: HepG2 and MCF7 cells. IHC-Fr: Human kidney tissue
  • General notes

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab81321 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
WB 1/1000 - 1/2000. Predicted molecular weight: 103 kDa.Can be blocked with Neuropilin 1 peptide (ab189308).
IHC-P 1/100 - 1/400. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

IP 1/100.
Flow Cyt 1/50 - 1/70.

The epitope that the antibody recognizes is intracellular. Fixation and permeabilization are necessary.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-Fr Use at an assay dependent concentration.
ICC/IF 1/250.

Target

  • Function
    The membrane-bound isoform 1 is a receptor involved in the development of the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits and in organogenesis outside the nervous system. It mediates the chemorepulsant activity of semaphorins. It binds to semaphorin 3A, The PLGF-2 isoform of PGF, The VEGF-165 isoform of VEGF and VEGF-B. Coexpression with KDR results in increased VEGF-165 binding to KDR as well as increased chemotaxis. It may regulate VEGF-induced angiogenesis.
    The soluble isoform 2 binds VEGF-165 and appears to inhibit its binding to cells. It may also induce apoptosis by sequestering VEGF-165. May bind as well various members of the semaphorin family. Its expression has an averse effect on blood vessel number and integrity.
  • Tissue specificity
    The expression of isoforms 1 and 2 does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules.
  • Sequence similarities
    Belongs to the neuropilin family.
    Contains 2 CUB domains.
    Contains 2 F5/8 type C domains.
    Contains 1 MAM domain.
  • Cellular localization
    Secreted and Cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • A5 protein antibody
    • BDCA4 antibody
    • BLOOD DENDRITIC CELL ANTIGEN 4 antibody
    • CD304 antibody
    • Neuropilin-1 antibody
    • Neuropilin1 antibody
    • NP1 antibody
    • NPN1 antibody
    • NRP 1 antibody
    • NRP antibody
    • NRP1 antibody
    • NRP1_HUMAN antibody
    • transmembrane receptor antibody
    • Vascular endothelial cell growth factor 165 receptor antibody
    • VEGF165R antibody
    see all

Images

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

    Lane 1 : Mouse heart tissue lysate
    Lane 2 : Rat heart tissue lysate

    Lysates/proteins at 20 µg per lane.

    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
    why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • The expression of Neuropilin 1 and the mesothelial marker cytokeratin was analyzed in human peritoneal specimens by immunohistochemistry. Positive cells for antibodies used (Neuropilin 1 (ab81321) and Cytokeratin) show brown staining. Nuclei are counterstained in blue. (a, b) Control peritoneal tissue, with a conserved mesothelial cell monolayer showing an epithelioid morphology (with a 20X objective). These cells show weak expression of Neuropilin 1 and a marked staining for cytokeratin (arrows). No expression of these proteins was observed in the submesothelial area (region under mesothelial monolayer) (c, d) Fibrotic tissue sample from peritoneal dialysis (PD) patient showing the loss of mesothelial monolayer and invading spindle-like mesothelial cells in submesothelial area (with a 40X objective). These cells present a strong staining for Neuropilin 1 (c), and are also positive for cytokeratin (d) (arrows). Pictures are representative of 5 cases of PD patient samples and 4 of control samples.

  • 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.

  • 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.

  • Unpurified ab81321 staining Neuropilin 1 in the COS1 fibroblast-like cell line derived from monkey kidney tissue by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% and blocked with 10% serum for 60 minutes at 24°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit monoclonal(1/500) was used as the secondary antibody.

    See Abreview

  • 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.

  • Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/10000 dilution (purified) + Human heart tissue lysate at 20 µg

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

    Predicted band size: 103 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • Unpurified ab81321 staining Neuropilin 1 in rat brain tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with paraformaldehyde and then blocked with 10% serum for 1 hour at 27°C followed by incubation with the primary antibody, undiluted, for 14 hours at 4°C. An undiluted Cy3® conjugated donkey anti-rabbit was used as the secondary antibody.

    See Abreview

  • ICC/IF image of unpurified ab81321 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab81321, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • 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.

  • Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/2000 dilution (purified) + Human placenta tissue lysate at 20 µg

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

    Predicted band size: 103 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?



    Blocking and dilution buffer: 5% NFDM/TBST.

  • 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).

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

    Lane 1 : Human placenta lysate
    Lane 2 : HUVEC cell lysate
    Lane 3 : HepG2 cell lysate
    Lane 4 : Mouse heart tissue lysate
    Lane 5 : Mouse kidney tissue lysate
    Lane 6 : Rat heart tissue lysate
    Lane 7 : Rat kidney tissue lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 103 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?

  • Unpurified ab81321 staining Neuropilin 1 in mouse brain tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 1 hour at room temperature; antigen retrieval was by heat mediation in citrate buffer (pH 6). Samples were incubated with primary antibody (1/100 in PBS + 2% blocking serum) for 16 hours at 4°C. A biotin-conjugated goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

    See Abreview

  • Immunohistochemical analysis of frozen human kidney tissue sections labelling Neurophilin 1 with ab81321 at a concentration of 1/100 for 18 hours at 4°C. The antibody was then blocked with a serum free protein block for 1 hour at 21°C. The secondary antibody used was a donkey anti-rabbit antibody conjugated to an Alexa488® dye inclubated at 1/400.

    See Abreview

References

This product has been referenced in:
  • Mahmoud M  et al. Smooth muscle cell-specific knockout of neuropilin-1 impairs postnatal lung development and pathological vascular smooth muscle cell accumulation. Am J Physiol Cell Physiol 316:C424-C433 (2019). Read more (PubMed: 30649916) »
  • Li H  et al. MicroRNA-320 targeting neuropilin 1 inhibits proliferation and migration of vascular smooth muscle cells and neointimal formation. Int J Med Sci 16:106-114 (2019). Read more (PubMed: 30662334) »
See all 49 Publications for this product

Customer reviews and Q&As

11-20 of 25 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100 0.5%
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 28 2012

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 10.0 Tris
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 2.5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 26 2012

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Spleen)
Specification
Spleen
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 16 2012

Application
Western blot
Sample
Human Cell lysate - whole cell (peripheral blood cells)
Loading amount
30 µg
Specification
peripheral blood cells
Gel Running Conditions
Reduced Denaturing (MES buffer, 4-12% Bis-Tris Gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 6% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 19 2012

Answer

Thank you for contacting us.
This is information would be interested in. We have not as yet tested the Anti-Neuropilin 1 antibody [EPR3113] (ab81321) ourselves in IHC wholemount. Please find the discount code and expiration date as requested below.
Discount code: xxxxxx
Expiration date: 28th December 2012
More information on the testing discount scheme can be found form the following link:
www.abcam.com/collaborationdiscount
If you have any further questions, please do not hesitate to ask.

Read More
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5µg/mL · Temperature: 25°C
Antigen retrieval step
None
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Permeabilization
Yes - 0.1% Triton X-100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 18 2012

Answer

Thank you once again for taking the time to complete our questionnaire and for your patience.

I have now heard back from our source, who have confirmed thatthey have tested Neuropilin-1 antibody on human placenta tissue, which has high expression of isoform 1 which indicates that this antibody can detect both isoforms.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been satisfactory. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I am pleased to offer you a free of charge replacement or credit note in compensation if you have purchased within the 6 month guarantee period. In order to process this, I would appreciate if you are able to provide the Abcam order reference number and date of purchase.

I can recommend you may also like to consider trying the following options:
1. antigen retrieval Try a stronger antigen retrieval eg EDTA
2. Try fresh samples.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10 mM Citrate pH 6.0
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 28 2012

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for IHC and human. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

As neuropilin 1 is expressed invascular tissue, we would expect this antibody to detect neuropilin 1 in the endothelial cells in vascular tissue. I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image from both the tissue typeswhich would help us to assess the results. Also, if you could describe how the samples were prepared, and if there were any differences.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.




Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which woudl help us to assess the results

Read More

Question
Answer

Thank you very much for your interest in ab71945 and ab81321.

To our knowledge, ab71945 and ab81321 have not been tested in zebrafish. Therefore, I can offer a discount off a future purchase if you buy ab71945 or ab81321now, test it inzebrafish and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab71945 or ab81321 in zebrafish. will then send a discount code. This code must be issued before purchasing ab71945 or ab81321 so please wait for my reply before ordering.

2. Purchase ab71945 or ab81321either by phone, fax, or online (www.abcam.com).

3. Test it in zebrafish.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for anyprimary antibodyordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab71945 or ab81321 turns out to be unsuitable for zebrafish, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

Read More

11-20 of 25 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up