MADCAM1 antibody (Middle Region)

Catalog No. ABIN3044076

The Rabbit Polyclonal anti-MADCAM1 antibody (ABIN3044076) specifically detects MADCAM1 in WB, IHC and IF. The antibody is reactive with Human samples. Independently validated for use in Immunohistochemistry.

Quick Overview for MADCAM1 antibody (Middle Region) (ABIN3044076)

Target: MADCAM1 (Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MADCAM1))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This MADCAM1 antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)

Product Details anti-MADCAM1 Antibody

Binding Specificity: AA 173-190, Middle Region

Purpose: Anti-MAdCAM1 Antibody

Sequence: EEPQGDEDVL FRVTERWR

Cross-Reactivity (Details): No cross-reactivity with other proteins

Characteristics: Anti-MAdCAM1 Antibody (ABIN3044076). Tested in IF, IHC, WB applications. This antibody reacts with Human. This is a premium antibody that guarantees superior quality, high affinity, and strong signals with minimal background in Western blot applications.

Purification: Immunogen affinity purified.

Immunogen: A synthetic peptide corresponding to a sequence in the middle region of human MAdCAM1.

Isotype: IgG

Application Details

Application Notes: Western blot, 0.1-0.5 μg/mL, Human
Immunohistochemistry (Paraffin-embedded Section), 2-5 μg/mL, Human
Immunofluorescence, 5 μg/mL, Human

Comment:

Antibody can be supported by chemiluminescence kit ABIN921124 in WB, supported by ABIN921231 in IHC(P) and IHC(F).

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #300031 (Immunohistochemistry)

by: Human Protein Atlas

No.: #300031

Date: 12/13/2016

Antigen: MADCAM1

Method validated: Immunohistochemistry

Positive Control: colon, spleen

Notes: Passed. ABIN3044076 staining is consistent with RNA-seq data and published literature.

Validation Images

Staining of human colon (A.) and spleen (B.) sections in a tissue microarray with ABIN3044076. See the protocol for detailed information.

Full Methods

Primary Antibody: ABIN3044076

Secondary Antibody: HRP polymer (ThermoFisher Scientific, TL-125-PH)

Full Protocol:

• Sample preparation:
• • Fixation and TMA preparation according to PMID: 22688270.
  • Cut 4µm TMA sections using a waterfall microtome (ThermoFisher Scientific, HM355S).
  • Dry sections ON at RT and then back them for 12-24h at 50°C.
• Deparaffinization and rehydration in xylene and graded alcohol series:
• • xylene twice for 3min.
  • 100% ethanol for 1min.
  • 95% ethanol for 1min.
  • 0.3% H₂O₂ in 95% ethanol for 5min to block endogenous peroxidase.
  • 70% ethanol for 1min.
  • 50% ethanol for 1min.
  • distilled H₂O for 2min.
• Antigen Retrieval by Heat Induced Epitope Retrieval (HIER):
• • Boil the sections in Lab Vision Citrate buffer pH6.0 (ThermoFisher Scientific, TA-250-PM1X) for 4min at 125°C in a decloaking chamber (Biocare Medical).
  • Allow slides to cool down to 90°C in the decloaking chamber.
• Immunostaining in a Lab Vision Autostainer 480 (ThermorFisher Scientific) at RT with 300µl of reagents for each step:
• • Rinse sections in Lab Vision wash buffer with extra tween added to a final concentration of 0.2% (ThermoFisher Scientific, TA-999-TT and TA-125-TW) wash buffer.
  • Block sections with Lab Vision Ultra V-Block (ThermoFisher Scientific, TA-125-UB) for 5min.
  • Rinse sections 2x in wash buffer.
  • Incubate sections with primary rabbit anti-MADCAM1 antibody (antibodies-online, ABIN3044076) diluted 1:900 for 30min.
  • Rinse sections 3x in wash buffer.
  • Incubate sections with labeled HRP polymer (ThermoFisher Scientific, TL-125-PH) for 30min.
  • Rinse sections 2x in wash buffer.
  • Develop in DAB Quanto solution (ThermoFisher Scientific, TL-125-QHDX) for 5min.
  • Rinse sections in distilled H₂O.
• Washing, counterstaining, and coverslipping in Autostainer XL (Leica Biosystems, ST5010) at RT:
• • Counterstaining in Mayer’s hematoxylin plus (HistoLab, 01820) for 7.5min.
  • Rinse sections in tap water for 5min.
  • Rinse sections in lithium carbonate water diluted 1:5 for 5min.
  • Dehydration in graded ethanol series and Neo-Clear (Merck Millipore, 109843) according to the manufacturer recommendations.
  • Automated mounting (Leica Biosystems, CV5030) of coverslip with Pertex (Histolab, 00871).
• Microscopy:
• • Image acquisition on a Leica Aperio AT2 and Aperio Scanscope AT at 20x magnification.

Experimental Notes: ABIN3044076 shows nice staining in a subset of vessels (endothelial cells), present in intestine and spleen.

Handling

Format: Lyophilized

Reconstitution: Add 0.2 mL of distilled water will yield a concentration of 500 μg/mL.

Concentration: 500 μg/mL

Buffer: Each vial contains 4 mg Trehalose, 0.9 mg NaCl and 0.2 mg Na2HPO4.

Handling Advice: Avoid repeated freezing and thawing.

Storage: 4 °C,-20 °C

Storage Comment: Store at -20°C for one year from date of receipt. After reconstitution, at 4°C for one month.
It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freeze-thaw cycles.

Expiry Date: 12 months

Target Details for MADCAM1

Target: MADCAM1 (Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MADCAM1))

Alternative Name: MADCAM1

Background:

Synonyms: Mucosal addressin cell adhesion molecule 1,MAdCAM-1,hMAdCAM-1,MADCAM1,

Tissue Specificity: Highly expressed on high endothelial venules (HEV) and lamina propia venules found in the small intestine, and to a lesser extent in the colon and spleen. Very low levels of expression found in pancreas and brain. Not expressed in the thymus, prostate, ovaries, testis, heart, placenta, lung, liver, skeletal muscle, kidney or peripheral blood leukocytes. .

Background: MADCAM1 (Mucosal Vascular Addressin Cell Adhesion Molecule 1), also known as MACAM1, is a protein that in humans is encoded by the MADCAM1 gene. By PCR-based analysis of somatic cell hybrids, Leung et al. (1997) mapped the MACAM1 gene to chromosome 19. The protein encoded by this gene is an endothelil cell adhesion molecule that interacts preferentially with the leukocyte beta7 integrin LPAM-1 (alpha4 / beta7), L-selectin, and VLA-4 (alpha4/beta1) on myeloid cells to leukocytes into mucosal and inflamed tissues. It is a member of the immunoglobulin superfamily and is similar to ICAM-1 and VCAM-1.

Molecular Weight: 60 kDa

UniProt: Q13477