| 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. The Laminin BioAssay(TM) ELISA Kit is a sandwich ELISA quantitative detection kit for human Laminin in cell culture supernates, cell lysates and serum. Detection Range: 156pg/ml-10,000pg/ml Sensitivity: <10pg/ml Specificity: Recognizes natural and recombinant human Laminin. There is no detectable cross-reactivity with other relevant proteins. Principle of the Assay: The Laminin BioAssay(TM) ELISA Kit is a solid-phase immunoassay specially designed to measure Human Laminin with a 96-well strip plate that is pre-coated with antibody specific for Laminin. The detection antibody is a biotinylated antibody specific for Laminin. The capture antibody is polyclonal antibody from rabbit and the detection antibody is polyclonal antibody from rabbit. The kit includes Human Laminin protein as standards with immunogen: Expression system for standard: from human fibroblasts. To measure human Laminin, add standards and samples to the wells, then add the biotinylated detection antibody. Wash the wells with PBS or TBS buffer, and add Avidin-Biotin-Peroxidase Complex (ABC-HRP). Wash away the unbounded ABC-HRP with PBS or TBS buffer and add TMB. TMB is an HRP substrate and will be catalyzed to produce a blue color product, which changes into yellow after adding the acidic stop solution. The absorbance of the yellow product at 450nm is linearly proportional to Human Laminin in the sample. Read the absorbance of the yellow product in each well using a plate reader, and benchmark the sample wells readings against the standard curve to determine the concentration of human Laminin in the sample. Kit Components: 143945A: Microtiter Strips, 1x96 wells *143945B: Laminin Standard, 2x10ng 143945C: Anti-human Laminin (Biotin), 1x100ul (1:100 dilution) 143696: Avidin-Biotin-Peroxidase Complex (ABC), 1x100ul (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 143702: Wash Buffer, 25X, 1x20ml Storage and Stability: Store *143945B powder at 4C. Once reconstituted store at 4C for up to 12 hours or at -20C for up to 48 hours. Store other components at 4C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Assay Summary: 1. Add 100ul of samples and standards and incubate the plate at 37C for 90 minutes or at RT for 2 hours. Do not wash. 2. Add 100ul biotinylated antibody and incubate the plate at 37C for 60 minutes or at RT for 90 minutes. Wash plate 3 times with Working Wash Buffer. 3. Add 100ul of ABC working solution and incubate the plate at 37C for 30 minutes or at RT for 40 minutes. Wash plate 5 times with Working Wash Buffer. 4. Add 90ul of TMB color developing agent and incubate the plate at 37C in dark for 15-25 minutes or at RT for 30 minutes. 5. Add 100ul TMB Stop Solution and read. |
