Product Uses Include
Laminin-1 is purified from EHS tumor tissue and is free of the laminin binding protein entactin which is a common contaminant in some laminin preparations (150 kDa). Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. The laminin is >90% pure (Figure 1).
The protein is modified to contain covalently linked HiLyte 488TM dyes (3) at random surface lysines. An activated ester of HiLyte 488TM is used to label the protein. Labeling stoichiometry is determined by spectroscopic measurement of protein and dye concentrations. Final labeling stoichiometry is 2-5 dyes per protein molecule (Figure 2). The material is guaranteed to contain <15% of free dye and >85% of dye conjugated to laminin. HiLyte 488TM laminin can be detected using a filter set of 502nm excitation and 527nm emission.
Laminin runs as individual subunits on SDS-PAGE with an apparent molecular weight of 400 and 225 kDa (Figure 1). LMN02 is supplied as an off white lyophilized powder. Each vial of LMN02 contains 20 µg protein.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >90% pure.
Fluorescent Fibronectin Treated MCF10A cells
Fluorescent fibronectin (Cat. # FNR01) treated MCF10Acells (image kindly provided by A. Varadara and M. Karthykenyan, Univ. S.Carolina,Columbia, SC).
Figure 1: HiLyte 488TM Laminin Purity Determination
Legend: 20 µg of unlabeled laminin (Lane 1) and 20 µg of HiLyte 488TM laminin (Lane 2) was separated by electrophoresis in a 4-20% SDS-PAGE system. The unlabeled protein was stained with Coomassie Blue and visualized in white light. The labeled protein was visualized under UV light. The alpha subunit runs at 400 kDa (top band) while the beta and gamma subunits run as a 225 kDa doublet (lower band). Arrows indicate unincorporated dye. In this example unincorporated dye = 13%. Protein quantitation was determined with the Precision Red™ Protein Assay Reagent (Cat. # ADV02). Mark12 molecular weight markers are from Invitrogen.
Figure 2: Absorption scan of HiLyte 488TM laminin in solution
Legend: LMN02 was diluted with Milli-Q water and its absorbance spectrum was scanned between 250 and 750 nm. In this example, HiLyte 488TM labeling stoichiometry was calculated to be 3.5 dyes per laminin protein using the absorbancy maximum for HiLyte 488TM at 527 nm and the Beer-Lambert law. Dye extinction coefficient when protein bound is 70,000M-1cm-1 .
1. Kelly T. et al. 1994. Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins. J. Cellular Physiol. 158: 299-308.
2. Tronchin G. et al. 1997. Expression and identification of a laminin-binding protein in Aspergillus fumigates conidia. Infection & Immunity 65: 9-15.
3. Use of this product employs the following patent rights licensed to Cytoskeleton, Inc. from Anaspec, Inc.: (a) the claims of U.S. Patents No. 7,754,893, 7,820,783 and 7,790,394; (b) any claims issuing from U.S. Patent Applications Serial No. 12/804,065, 12/807,268 and 12/925,505; and © all patents to be issued pursuant thereto, and all continuations, continuations –in-part, reissues, substitutes, and extensions thereof. The use of this product is limited to the field of use comprising internal use by an end user of this product solely in in vivo and in vitro cell staining or biochemical assay applications, such as IHC, HCS, FACS, in vitro assays of an end user only for scientific R&D purposes. The filed of use of this product explicitly excludes the following actions: (a) generating data from clinical applications in humans and animals; and (b) generating QC or QA data for the validation of health, food or cosmetic products.
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Question 1: What is the optimal excitation and emission filter settings to visualize the HiLyte Fluor™ 488 fluorescence?
Answer 1: HiLyte Fluor™ 488 labeled-laminin can be detected using a filter set of 502 nm excitation and 527 nm emission.
Question 2: What is the labeling stoichiometry?
Answer 2: HiLyte 488™ labeling stoichiometry was calculated to be 2-5 dyes per laminin protein using the absorbancy maximum for HiLyte 488™ at 527 nm and the Beer-Lambert law. Dye extinction coefficient when protein bound is 70,000 M-1cm-1.
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