MA5-29413

antibody from Invitrogen Antibodies

Targeting: MCAM CD146, HEMCAM, MelCAM, METCAM, MUC18

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Data presented on provider website)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-29413

Provider

Invitrogen Antibodies

Product name

CD146 Recombinant Rabbit Monoclonal Antibody (5)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

This product is preservative free. It is recommended to add sodium azide to avoid contamination (final concentration 0.05%-0.1%). Recombinant rabbit monoclonal antibodies are produced using in vitro expression systems. The expression systems are developed by cloning in the specific antibody DNA sequences from immunoreactive rabbits. Then, individual clones are screened to select the best candidates for production. The advantages of using recombinant rabbit monoclonal antibodies include: better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity to diverse targets due to larger rabbit immune repertoire. This antibody has specificity for Human CD146/MCAM.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

5

Vial size

100 µL

Concentration

1 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

Submitted references

Wnt5a promotes renal tubular inflammation in diabetic nephropathy by binding to CD146 through noncanonical Wnt signaling.

Li X, Wen J, Dong Y, Zhang Q, Guan J, Liu F, Zhou T, Li Z, Fan Y, Wang N
Cell death & disease 2021 Jan 18;12(1):92

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of CD146 in Lane A: Hela Whole Cell Lysate (30 µg). Samples were probed using a CD146 Monoclonal Antibody (Product # MA5-29413) at a 1:500 dilution, followed by a Goat Anti-Rabbit IgG (H+L), Dylight 800 Secondary Antibody at a 1:10000 dilution. Western blot was performed under reducing conditions. Predicted band size:72 kDa. Observed band size:115 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot using CD146 Recombinant Rabbit Monoclonal Antibody (5) (Product # MA5-29413) at 1:500 dilution. Lane A: Hela Whole Cell Lysate. Lysates/proteins at 30 μg per lane. Secondary Goat Anti-Rabbit IgG H&L (DyLight™ 800) at 1:10,000 dilution. Developed using the Odyssey technique. Performed under reducing conditions. Predicted band size: 72 kDa. Observed band size: 115 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-CD146 Recombinant Rabbit Monoclonal Antibody (5) (Product # MA5-29413) and a 130 kDa band corresponding to Cell surface glycoprotein MUC18 was observed across cell lines. Whole cell extracts (30 µg lysate) of SK-MEL-31 (Lane 1), SK-BR-3 (Lane 2), HUVEC (Lane 3), HeLa (Lane 4), T-47D (Lane 5), MCF7 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Cell surface glycoprotein MUC18 was performed using 70% confluent log phase SK-MEL-28 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with CD146 Recombinant Rabbit Monoclonal Antibody (5) (Product # MA5-29413) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained withRhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Membrane localization. Panel e represents Low expression in MCF-7. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 1 The expression levels of Wnt5a and CD146 were upregulated in diabetic neuropathy (DN) patients. A Representative immunostaining of Wnt5a and CD146 on normal kidney sections of nephrectomy samples ( n = 5) and kidney biopsy samples of patients with minimal change disease (MCD) ( n = 5) and DN ( n = 15). Original magnification, x200; scale bar, 100 mum. B Quantitative analysis of Wnt5a staining in kidney sections from each group according to the average integrated optical density (IOD). *** P < 0.001. C Quantitative analysis of expression of CD146 staining on kidney sections of each group by the average IOD. *** P < 0.001. D , E The concentration of soluble CD146 (sCD146) in serum and urine samples of each group. *** P < 0.001. Immunostaining was performed in duplicate. Significant differences were determined by unpaired two-tailed t -tests.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 2 Assessment of renal inflammation in human kidney biopsies. A Immunostaining using specific antibodies for different immune cells, including T lymphocytes (CD3 + cells), B lymphocytes (CD20 + cells) and monocytes/macrophages (CD68 + cells), was performed on normal subjects, MCD patients, and DN patients. Original magnification, x400; scale bar, 50 mum. Immunohistochemical staining of the proinflammatory marker, chemokine (C-C motif) ligand 2 (CCL-2) was also performed. Original magnification, x200; scale bar, 50 mum. B - D The numbers of CD3 + , CD20 + , and CD68 + cells per high-power field (HPF) were counted in the tubulointerstitium. *** P < 0.001, * P < 0.05. E Quantitative analysis of the expression of CCL-2 staining on kidney sections of each group according to the average IOD. ** P < 0.01. Immunostaining was performed in duplicate. Significant differences were determined by unpaired two-tailed t -tests. F Correlation of Wnt5a and CD146 expression with inflammatory process and known prognostic factors (percentage of IFTA, renal function, and proteinuria) in patients with diabetic nephropathy. Data are r value based on Pearson and Spearman correlation analysis; ( P value); NS is not significant. IFTA, interstitial fibrosis and tubular atrophy; Estimated GFR, Estimated glomerular filtration rate; HbA1c, hemoglobin A1c.