Laminin BioAssay(TM) ELISA Kit, Human

Sandwich ELISA quantitative detection kit for Human Laminin in cell culture supernates, serum and plasma (heparin, EDTA). Laminin is a large basement membrane glycoprotein composed of three subunits designated the A, B1, and B2.1 Laminin has diverse biological functions, which include stimulating epithelial cell growth and differentiation. The nucleotide sequence of human laminin A chain has an open reading frame encoding 3075-amino acids. The human laminin A chain is at locus 18p11.3.3 The nucleotide sequence of the human laminin B1 reveals a 5358-base pair open reading frame that potentially codes for 1786 amino acids, including 20 amino acids of a presumptive signal peptide. The gene for the human laminin-B1 chain has been localized to chromosome 7, band q31. The B2 chain consists of six distinct domains, including two domains with alpha-helical, coiled-coil structures, two domains with cysteine-rich homologous repeats, and two globular domains. The amino acid sequences of the B2 and B1 chains demonstrate considerable homology. The human laminin B2 chain gene maps to the long arm of chromosome 1 in the band q31. , Assay Range:, 156pg/ml-10,000pg/ml, Sensitivity: < 10 pg/ml, Crossreactivity: There is no detectable cross-reactivity with other relevant proteins. Uniprot ID: B0YJ33, Entrez Gene ID: 3909, Principle of the Assay: The Human Laminin ELISA Kit is based on standard sandwich enzyme-linked immunosorbent assay technology. A rabbit polyclonal antibody specific for Laminin has been pre-coated onto 96-well plates. Standards (from human fibroblasts) and test samples are added to the wells, incubated and then biotinylated detection polyclonal antibody from rabbit specific for Laminin is added, followed by an incubation step and a washing step with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. HRP substrate TMB is used to visualize the HRP enzymatic reaction. TMB is catalyzed by HRP to produce a blue colored product that changes into yellow upon addition of acidic stop solution. The intensity of the yellow color is proportional to the amount of Laminin in the sample. Kit Components: 143945A: Microtiter Plate, 1x96wells. Pre-coated with anti-human Laminin antibody, 143945B: Laminin standard, 2x10ng, 143945B: Anti-human Laminin (Biotin), 1x130ul (1:100 dilution), 143696C: Avidin-Biotin-Peroxidase Complex (ABC), 1x130ul (1:100 dilution), 143697: Sample Diluent Buffer, 1x30ml, 143698: Antibody Diluent Buffer, 1x12ml, 143699: ABC Diluent Buffer, 1x12ml, 143700: TMB Color Developing Agent, 1x10ml, 143701: TMB Stop Solution, 2N H2SO4, 1x10ml, Storage and Stability: Unopened kit is stable at 4°C for 6 months. Material Required But Not Provided: 1. Microplate reader in standard size, 2. Automated plate washer, 3. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended when a large number of samples are to be assayed , 4. Clean tubes and Eppendorf tubes, 5. Washing buffer (neutral PBS or TBS), Assay Summary: 1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash. , 2. Add biotinylated antibodies and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS or PBS., 3. Add AB Cworking solution and incubate the plate at 37°C for 30min. Wash plate 5times with 0.01M TBS or PBS., 4. Add TMB color developing agent and incubate the plate at 37°C in dark for 15-20 min., 5. Add Stop solution and read absorbance at 450nm.