Anti-Laminin-5 (γ2 chain) Antibody, clone D4B5

Laminins are basement membrane, extracellular matrix glycoproteins that are composed of alpha, beta and gamma heterotrimeric chains. Different combinations of five alpha chains, three beta chains, and two gamma chains form at least 12 laminin isoforms. Laminin-5 (LN5), which consists of laminin alpha3, beta3 and gamma2 chains, is a highly conserved component of anchoring filaments underlying the hemidesmosome structure of epidermal keratinocytes. Mutations or deletion in the LN5 genes (LAMA3, LAMB3, and LAMC2) is associated with epidermolysis bullosa, a lethal skin blistering disease. LN5 may stabilize the epidermal/dermal junction of skin through binding with integrins alpha3/beta1, alpha6/beta4 and type VII collagen and has a critical role in the anchorage and locomotion of a wide variety of cell types, including epithelial cells, fibroblasts, neurons and leukocytes. Compared to fibronectin, collagen, or vitronectin, cells of epithelial origin will adhere to Laminin-5 faster and will spread to a larger extent. Recently, Laminin-5 is attracting attention as a substrate for carcinoma cells that may stimulate migration, since it has been detected at the leading edge of invasive cancer tissue in several types of human carcinomas (Niki, et al., 2002, Lohi, et al., 2001).

Monoclonal antibody MAB19562 was developed against amino acid residues 382-608 of human laminin-5 gamma2 chain (III domain) expressed as a GST fusion protein. The antibody recognizes the 150 (precursor form) and 105 kDa (mature form) chain proteins of Laminin-5. No reactivity is expected with other laminin types, as the gamma2 chain of laminin is unique to laminin-5.

Amino acid residues 382-608 of human laminin-5 gamma2 chain (III domain) expressed as a GST fusion protein

Anti-Laminin-5 (γ2 chain) Antibody, clone D4B5 is an antibody against Laminin-5 (γ2 chain) for use in ELISA, IH(P) & WB.

Research Category
Cell Structure

Research Sub Category
ECM Proteins

Western blot: 5ug/ml is recommended; dilute antibody in PBS or TBS only, not blocking solutions; blocking with purified compounds is suggested.

Immunohistochemistry of paraffin sections: 5-10ug/ml is suggested. Do not over fix. A light 4% or 2% PFA are best. 4 mm thick paraffin sections are dewaxed, rehydrated and immersed in 0.3% hydrogen peroxide-containing methanol for inactivation of intrinsic peroxidase, followed by treatment with Protease XXIV (Sigma) for 15 min at room temperature (0.01-0.05%). Do not use trypsin. Antibody incubation at 37°C for 1 hour, followed by secondary antibody detection and visualization with DAB (Mizushima et al. 1998). Do not place the monoclonal antibody in serum blocks because serum conatins laminin 5 which will bind and interfere with the antibody. Use BSA or other such media instead.

Strongly positive staining is seen in Basement Membrane of the following tissues: Skin, Esophagus, Lung, and Kidney. Positive staining is also seen in Stomach, Small intestine, Spleen, Thymus, Prostate, and Ovary. Thyroid, Testis, Skeletal and Cardiac muscle were negative for LN5 staining.

Inhibition of alpha2beta1 integrin binding to laminin 5 (40ug/ml) (Decline & Rousselle 2000).

ELISA Optimal working dilutions must be determined by end user.

Evaluated by Western Blot on recombinant Laminin-5.

Western Blot Analysis: A 1:500-1:2000 dilution of this antibody detected Laminin-5, gamma2 chain in 10 µg of human recombinant Laminin-5.

150 kDa (precursor form) and 105 kDa (mature form)

Format: Purified

Protein A Purified mouse immunoglobulin in 20 mM sodium phosphate, 250 mM NaCl, pH. 7.6, with 0.1% sodium azide as a preservative.

Protein A purified

Maintain for 1 year at 2–8°C from date of shipment. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Control
Human pancreatic tumor tissue, human breast carcinoma tissue or A-431 whole cell lysate

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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