MCAM/CD146 Antibody [Unconjugated]

Western blot shows lysates of Bowes human melanoma cell line and mouse embryo tissue. PVDF Membrane was probed with 1 µg/mL of Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for MCAM/CD146 at approximately 117 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

MCAM/CD146 was detected in immersion fixed B16-F1 mouse melanoma cell line using Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to plasma membranes. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Simple Western lane view shows lysates of mouse embryo tissue (15 d.p.c.), loaded at 0.2 mg/mL. A specific band was detected for MCAM/CD146 at approximately 138 kDa (as indicated) using 10 µg/mL of Sheep Anti-Mouse MCAM/CD146 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6106) followed by 1:50 dilution of HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Perivascular cells and subaortic mesenchyme are the main source of BMPER within the AGM region. (A) Distribution of BMPER protein in a transverse section of E10.5 AGM measured by immunostaining. Green, CD31; magenta, BMPER; cyan, RUNX1; blue, DAPI. gut, hindgut; nc, notochord. Bar, 50 µm. (B) Bmper mRNA in transverse section of the E10.5 AGM region by in situ hybridization. Bar, 50 µm. (C) Quantification of the mean immunostaining signal intensity (mean gray values) of DAPI (blue) and BMPER (magenta) along a box (not depicted) drawn over the dorsal-ventral axis of the AGM region from A. Distance is from the notochord (dorsal), position 0, to the intersection with the gut and the AGM region (ventral), position 500. (D) Higher magnification of the highlighted region from A showing the aortic endothelium and perivascular population. Green, CD31; magenta, BMPER; cyan, RUNX1; blue, DAPI. Bar, 50 µm. (E) Higher magnification of the region highlighted in B showing Bmper mRNA around the lining of the aorta. Bar, 50 µm. (F) Expression level of Bmper relative to Tbp in each sorted population: Lin−VC−CD45+, representing hematopoietic cells; Lin−VC+CD45−, endothelial cells; Lin−VC−CD45−CD146+, putative perivascular cells; and Lin−VC−CD45−CD146−, remaining stroma. Expression was normalized to the Lin−VC−CD45−CD146+ population. Each population as percentage of Lin− cells indicated below. Sorting was performed twice, first from one pool of embryos from four to five litters and second from two pools of embryos from four to five litters. Error bars represent SD from the mean (n = 3). Significance calculated by t test: **, P = 0.0016; ***, perivascular versus stroma, P = 0.0006; perivascular versus hematopoietic, P = 0.0002. (G) The distribution of nonendothelial, CD146-positive cells and endothelial CD146-positive cells in transverse section of the E10.5 AGM region. Green, CD31; red, CD146; blue, DAPI. Bar, 50 µm. (H) Higher magnification view of the region highlighted in G showing CD31- and CD146-positive endothelial layer and the CD146-positive CD31-negative perivascular layer around the dorsal aorta. Green, CD31; red, CD146; blue, DAPI. Bar, 50 µm. (I) Expression level of Bmper relative to Tbp in each sorted population: Lin−VC+CD45−CD43−CD41lo proHSC, Lin−VC+CD43+CD41+ type I preHSC, and Lin−VC+CD45+ type II preHSC and Lin−VC−CD45−CD146+ putative perivascular cells. Each population as percentage of Lin− cells indicated below. Sorting was performed twice, from pools of two and six litters, respectively. Expression was normalized to the Lin−VC−CD45−CD146+ population. Error bars represent SD from the mean (n = 2). Significance calculated by t test: *, perivascular versus preHSCI, P = 0.04; perivascular versus preHSCII, P = 0.03. (J) Higher magnification view of highlighted region from A showing the localization of BMPER protein in the hematopoietic clusters of the E10.5 dorsal aorta in sections measured by immunostaining. Magenta, BMPER; green, CD31; cyan, RUNX1; blue, DAPI. Bar, 50 µm. (K and L) Higher magnification view of highlighted region from B (K) and from Fig. S3 C (L) showing Bmper mRNA in some but not all cells of the intra-aortic cluster by in situ hybridization. Bars, 50 µm. Image collected and cropped by CiteAb from the following publication (//rupress.org/jem/article/214/12/3731/42286/A-molecular-roadmap-of-the-AGM-region-reveals), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Mu
• Applications: WB, Simple Western, ICC/IF
• Clonality: Polyclonal
• Host: Sheep
• Conjugate: Unconjugated
• Concentration: LYOPH

MCAM/CD146 Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant mouse MCAM/CD146
  Val24-Tyr563
  Accession # Q8R2Y2
• Specificity: Detects mouse MCAM/CD146 in direct ELISAs and Western blots. In direct ELISAs, approximately 40% cross‑reactivity with recombinant rat MCAM is observed and approximately 10% cross-reactivity with recombinant human MCAM is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Sheep
• Gene: MCAM
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 5-15 ug/mL
  • Simple Western 10 ug/mL
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for MCAM/CD146 Antibody [Unconjugated]

• CD146 antigen
• CD146
• cell surface glycoprotein MUC18
• Cell surface glycoprotein P1H12
• L-Gicerin
• MCAM
• melanoma adhesion molecule
• melanoma cell adhesion moleculeS-endo 1 endothelial-associated antigen
• Melanoma-associated antigen A32
• Melanoma-associated antigen MUC18
• MUC18
• MUC18Gicerin

Background

MCAM (Melanoma cell adhesion molecule; also CD146 and MUC18) is a 110‑120 kDa member of a small group of Ig‑superfamily molecules that includes CD239 and CD166. MCAM has also been reported at a molecular weight of approximately 150 kDa. In rodent, MCAM is reportedly expressed on neurons, endothelial cells, NK cells, neutrophils, mesenchymal stem cells and melanoma cells. MCAM appears to contribute to intercellular endothelial cell junctions, and possibly contributes to the migration of select cell types. Mature mouse MCAM is a 625 amino acid (aa) type I transmembrane glycoprotein. Its extracellular region is 540 aa in length (aa 24‑563). It contains two V‑type Ig‑like domains (aa 24‑244) followed by three C2‑type Ig‑like domains (aa 246‑512). One cytoplasmic region splice form shows a seven aa substitution for aa 600‑648. Unlike human, rodent MCAM does not undergo a splicing event that will generate a soluble isoform. Over aa 24‑563, mouse MCAM shares 90% and 74% aa identity with rat and human MCAM, respectively.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for MCAM/CD146 Antibody (AF6106)(1)

Publication using AF6106

• Alison C. McGarvey, Stanislav Rybtsov, Céline Souilhol, Sara Tamagno, Ritva Rice, David Hills et al. A molecular roadmap of the AGM region reveals BMPER as a novel regulator of HSC maturation Journal of Experimental Medicine 2017-12-04 [PMID: 29093060] (Immunohistochemistry, Mouse)

Review for MCAM/CD146 Antibody (AF6106) (1)

Average Rating: 4

(Based on 1 review)

We have 1 review tested in 1 species: Mouse.

reviewed by: Verified Customer IHC-Fr Mouse 01/08/2015

Summary

• Application: Immunohistochemistry-Frozen
• Sample Tested: See PMID 22699974
• Species: Mouse

Bioinformatics

• Gene Symbol: MCAM
• Uniprot:
  • Q8R2Y2()