Overview

  • Product name
    Anti-CD146 antibody [EPR3208]
    See all CD146 primary antibodies
  • Description
    Rabbit monoclonal [EPR3208] to CD146
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human CD146 aa 600 to the C-terminus.
    Database link: P43121

  • Positive control
    • WB: A375, HUVEC and B16-F0 cell lysate; Human fetal artery lysate and Rat placenta ICC/IF: Murine bone marrow cell lysates. IHC-Fr: Mouse spleen tissue. IHC-P: Melanoma, breast carcinoma vessel, urinary bladder transitional carcinoma vessel, glioma vessel, normal tonsil and normal spleen tissue. FC: A375 and HUVEC cells
  • 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab75769 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
ICC/IF 1/100 - 1/250.
IHC-Fr 1/250.
WB 1/1000. Predicted molecular weight: 72 kDa.Can be blocked with CD146 peptide (ab219161).

For unpurified use at 1/10000 - 1/50000.

IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Flow Cyt 1/20 - 1/80.

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

Target

  • Function
    Plays a role in cell adhesion, and in cohesion of the endothelial monolayer at intercellular junctions in vascular tissue. Its expression may allow melanoma cells to interact with cellular elements of the vascular system, thereby enhancing hematogeneous tumor spread. Could be an adhesion molecule active in neural crest cells during embryonic development. Acts as surface receptor that triggers tyrosine phosphorylation of FYN and PTK2, and a transient increase in the intracellular calcium concentration.
  • Tissue specificity
    Detected in endothelial cells in vascular tissue throughout the body. May appear at the surface of neural crest cells during their embryonic migration. Appears to be limited to vascular smooth muscle in normal adult tissues. Associated with tumor progression and the development of metastasis in human malignant melanoma. Expressed most strongly on metastatic lesions and advanced primary tumors and is only rarely detected in benign melanocytic nevi and thin primary melanomas with a low probability of metastasis.
  • Sequence similarities
    Contains 3 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 2 Ig-like V-type (immunoglobulin-like) domains.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • A32 antigen antibody
    • CD 146 antibody
    • CD146 antibody
    • CD146 antigen antibody
    • Cell surface glycoprotein MUC18 antibody
    • Cell surface glycoprotein P1H12 antibody
    • Gicerin antibody
    • Mcam antibody
    • Melanoma adhesion molecule antibody
    • Melanoma associated antigen A32 antibody
    • Melanoma associated antigen MUC18 antibody
    • Melanoma associated glycoprotein MUC18 antibody
    • Melanoma cell adhesion molecule antibody
    • Melanoma-associated antigen A32 antibody
    • Melanoma-associated antigen MUC18 antibody
    • MelCAM antibody
    • MUC 18 antibody
    • MUC18 antibody
    • MUC18_HUMAN antibody
    • S endo 1 antibody
    • S endo 1 endothelial associated antigen antibody
    • S-endo 1 endothelial-associated antigen antibody
    see all

Images

  • Immunohistochemistry experiments were used to compare symptomatic carotid plaques (SC) and asymptomatic carotid plaques (AsC)

    Asymptomatic lesions presented higher CD146+ pericyte infiltration, p<0.001. Representative images are on the left with corresponding quantification on the right.

    ab75769 used at 1/200 dilution.

    (After Figure 2 of Davaine et al)

  • Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
    Lane 2: CD146 knockout HAP1 whole cell lysate (40 µg)
    Lane 3: A375 whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab75769 observed at 120-72 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab75769 was shown to specifically react with CD146  in wild-type HAP1 cells as signal was lost in CD146 knockout cells. Wild-type and CD146 knockout samples were subjected to SDS-PAGE. ab75769 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD146 with purified ab75769 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Flow Cytometry analysis of A375 (human malignant melanoma) cells labeling CD146 with unpurified ab75769 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

  • Immunocytochemistry/Immunofluorescence analysis of A375 (human malignant melanoma) cells labelling CD146 with purified ab75769 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 (1/1000) were also used.

    Control 1: primary antibody (1/100) 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-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified)

    Lane 1 : A375 cell lysate
    Lane 2 : Human fetal artery lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 72 kDa



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

  • Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + HUVEC cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 72 kDa



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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of melanoma tissue labelling CD146 with unpurified ab75769 at 1/250. A HRP/AP polymerized secondary antibody was used.

  • Immunohistochemistry (Frozen sections) analysis of mouse spleen tissue labelling CD146 with unpurified ab75769 at 1/250. Tissue samples were fixed with acetone and blocked with 5% serum for 2 hours at 25°C. The sample was incubated with primary antibody at 4°C for 12 hours. An Alexa Fluor®488-conjugated Goat polyclonal to rabbit IgG (1/250) was used as secondary antibody. Nuclear staining was with DAPI (blue).

    See Abreview

  • Flow Cytometry analysis of HUVEC cells labelling CD146 with purified ab75769 at 1/50 (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-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + B16-F0 cell lysate at 20 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 72 kDa



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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast carcinoma vessels tissue labelling CD146 with unpurified ab75769.

  • Anti-CD146 antibody [EPR3208] (ab75769) at 1/10000 dilution (purified) + Rat placenta lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 72 kDa



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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of urinary bladder transitional carcinoma vessels tissue labelling CD146 with unpurified ab75769.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of glioma vessels tissue labelling CD146 with unpurified ab75769.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal tonsil tissue labelling CD146 with unpurified ab75769.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal spleen tissue labelling CD146 with unpurified ab75769.

References

This product has been referenced in:
  • Iida M  et al. A sialo-oligosaccharide-rich mucin-like molecule specifically detected in the submandibular glands of aged mice. Arch Oral Biol 97:52-58 (2019). Read more (PubMed: 30343214) »
  • Ho TC  et al. PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells. Stem Cell Res Ther 10:2 (2019). Read more (PubMed: 30606221) »
See all 57 Publications for this product

Customer reviews and Q&As

1-10 of 13 Q&A

Answer

This is the Angiopoietin-1, its target being the interstitial stem cells destined to become microvascular pericytes. The catalogue number is ab8451. This antibody works well with IHC and ICC.

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Answer



Please see below.

Anti-alpha smooth muscle Actin antibody (ab21027),

Anti-Desmin antibody [Y266] (ab32362),

Anti-SPARC antibody or osteonectin (ab14174)

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Answer

Thank you for your email.

Yes, the immunogen is from an intracellular region, thus the cells would need to be fixed and thenpermeabilized so that the antibody is able to get to the binding site.

If you did not permeabilize the HUVEC cells that would explain the results you are seeing.

I am sending you the link to our protocol for intracellular staining in flow cytometry:
https://www.abcam.com/index.html?pageconfig=resource&rid=11448



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.

The anibody has been tested in flow cytometry and would be guaranteed for this application.

The recommended starting dilution is 1/80 for flow cytometry.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for sending this information. It is very helpful.

The IF protocols used for the images on our website use 100% acetone or 100% methanol respectively and do not use any ethanol. As fixation is very often a cause for an antibody to not perform as it should I would recommend using acetone or methanol at -20C for 10 minutes followed by washing with PBS or PBS + 1% BSA. A primary antibody dilution of ˜1/250 should lead to some signal but you may wish to increase the concentration of the antibody as well to ensure reactivity.

Please let me know how these suggestions work. This product is covered by our Abpromise guarantee. If we cannot remedy this issue and this is a product that you have purchased within the last six months, we will replace or refund it under our Abpromise guarantee, as you are using it according to specifications listed on our datasheet.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Question
Answer

Thank you for contacting us.
The peptide maps between amino acids 600 - 646 of human CD146, near the C-terminus (UniProt: P43121). This corresponds to the intracellular region.
http://www.uniprot.org/uniprot/P43121
I hope this information helps. Please contact us with any other questions.

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Question
Answer

I now have some further information to share with you in regards to the likelihood of ab28364 and ab75769 detecting the canine protein.


Based on sequence analysis, theab75769 is not expected to cross react with canine protein (SwissProt reference F1P894)as there is onylthere is only 30% similarity between the immunogen used to raise the antibody and the canine protein. A similar result was found with ab28364 where only a 55% homology was found (with SwissProt reference E2R6C1).


However, we have not tested either of these antibodies with canine samples and as the SwissProt references I found have not been classified as validated, I would not like to say for definite either way whether the antibodies will detect the protein in canine samples or not. I would say as you have the antibodies in house, I would try them with your canine samples to see what this yields.You may however need to find alternative antibodies to use.


I hope this information has been of help. If I can help in any other way, please donot hesitate to contact me again.

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Question
Answer

Thank you for contacting us yesterday.

I have been looking into your query into if anti-CD31 (ab28364) and anti-CD146 antibody (ab75769) are likely to be able to detect the canine proteins. Let me first just say that it seems that the sequences of the canine proteins do not seem very well characterised to this point (none of the SwissProt references for the canine sequences mentioned in this email have been fully characterised). It is therefore difficult to draw strong conclusions on if the antibodies are likely to cross react or not.

The anti-CD31 you are using is raised against a peptide taken from the C-terminal end of the mouseCD31 protein. Through performing a sequence comparison of this sequence(SwissProt reference http://www.uniprot.org/uniprot/Q08481) to that of the canine protein (SwissProt reference http://www.uniprot.org/uniprot/E2R6C1), it only shares 76% homology overall and the regions of matching residues in the C-terminal is quite patchy. I am checking further to see exactly where the immunogen used with this antibody binds and will let you know as soon as I have this information.

If you find thatab28364 is not working in your staining of canine samples, unfortunately, we do not currently haveany anti-CD31 antibodieswhich have beenused with canine samples. Asthecanine proteinsequence has a higherhomology with thehuman protein (SwissProt reference http://www.uniprot.org/uniprot/P16284)than the mouse proteinIwould suggest testing an antibodyraised against thehuman protein would be more likely to be successful. If you would like me to go through the antibodies we have raised against the human protein to find the most appropriate one to try please do let me know.

The anti-CD146 (ab75769), looks somewhat more promising. The human sequence (SwissProt reference http://www.uniprot.org/uniprot/P43121, from which the immunogen is taken from), shares 87 % homology with the canine protein (SwissProt reference http://www.uniprot.org/uniprot/F1P894). However, the immunogen is taken from the intracellular domain (residues 584-646) and the canine protein, from this sequence at least, only goes up to 581 residues. I am again checking further to see what the exact immunogen used is and will let you know as soon as I have this information.

As discussed over the phone, if you find that ab75769 is not performing well with your canine samples, we do have a mouse monoclonalantibody, https://www.abcam.com/CD146-antibody-P1H12-ab24577.htmlwhich has been validated in dog. This antibody has however not been used with PFA fixed, paraffin embedded sections as yet. The same clone [P1H12] conjugated to biotin (https://www.abcam.com/CD146-antibody-P1H12-Biotin-ab77928.html)has however been used for this application so the likelyhood is that itwould work.If you would like to try ab24577 in IHC-P you would be eligible for a testing discount. This is a scheme which we run to gather more data about our antibodies by our customer.

This would involve you purchasing ab24577, testing it in IHC-P with you canine (or human samples) and letting us know of the results through an Abreview. This should only take 5-10 minutes.Whether the results are positive or negative, you would then be rewarded with a free primary antibody of your choice from our catalogue.The only constraint is that the antibody to be tested needs to be purchased and the Abreview submitted and the "free" antibody claimed within 4 months. More information on this scheme can be found from the following link:

https://www.abcam.com/collaboratordiscount

As requested I have also had a look into if we have any antibodies which would be suitable for use in detecting CD34 in your canine samples. We havea mouse monoclonalantibody which have been used with canine samples previously, https://www.abcam.com/CD34-antibody-1H6-ab35036.html. However, this antibody has as yet only been characterisedwith western blotting and flow cytometry. If you would be interested you would therefore again be eligible for a testing discount if youwere to purchase this and test it in IHC staining of paraffin embedded sections.

If you would be interested in this testing discount scheme, please do let me know as a discount code needs to be issued prior to purchasing the antibody to be tested.

I thought you might like some more information on the Abreview system as well. We encourage all our customers to review their experiences with our antibodies so that other customers can evaluate which antibodies would be best for them to use. As a reward for submitting reviews you earn Abpoints. These can be redeemed against future purchases, or exchanged for Amazon vouchers. More information on how the system works can be found from the following link:

https://www.abcam.com/abreviews

I hope this information has been of help. I will let you know as soon as I have further information to share on the immunogens used to raise ab28364 and ab75769.

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Answer

Thank you for contacting us.

Unfortunately we have not tested any of the anti-CD146 antibodies in pig species, therefore we cannot predict their cross reactivity with this species.

For antibody ab78488 we have not done any epitope mapping, and so we cannot make any guess as to whether the region that the antibody binds to will share homology with the pig protein.

For antibody ab75769, the peptide maps between amino acids XXXX - XXXX of human CD146, near the C-terminus (UniProt: P43121). I was not able to find the pig protein sequence to run a BLAST and check the alignment.

This is all the information available, I am sorry I could not be more helpful. For further assistance, please do not hesitate to contact us.

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Answer

Thank you for your enquiry regarding ab75769. Every product has specific storage information and I would advise your customer to follow the storage instructions printed on the product datasheet. If you need any further assistance in the future, please do not hesitate to contact me.

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1-10 of 13 Q&A

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