Microcrystalline wax (Ibercer 2616, melting point of 81 °C) was kindly provided by Iberceras Specialities S.L.U. (Madrid, Spain). We purchased 100 µm-thick polyester transparency A4 sheets from APLI S.L. (Alaquàs, Spain). Pressure-sensitive-adhesive films (PSA) (ARcare® 8939, 120 µm total thickness) were obtained from Adhesives Research Inc. (Glen Rock, PA, USA). Poly(methyl methacrylate) (PMMA) was acquired from Servicio Estación (Barcelona, Spain). Unbacked 120 µm-thick nitrocellulose was obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Polydimethylsiloxane (PDMS Sylgard 184 kit, which includes the Sylgard 184 silicone elastomer base and the Sylgard 184 silicone elastomer-curing agent) was purchased from Sigma Aldrich (St. Louis, MO, USA). For the immunoassay, the capture (anti-human) and detector antibodies (anti-human-HRP), along with the analyte (TNFα), were obtained from Sinobiological Inc. (Beijing, China). The buffer reagents (Phosphate Buffered Saline (PBS), Tween 20, Bovine Serum Albumin (BSA)), the dyes (Tartrazine, Erioglaucine and Allura red), the streptavidine-HRP, as well as the substrate (3,3′,5,5′-Tetramethylbenzidine (TMB)) were obtained from Sigma Aldrich (USA). Sodium Hydrogen Carbonate (NaHCO3) was purchased from Panreac Química (Barcelona, Spain).
Streptavidine hrp
Manufactured by Merck Group
Sourced in United States
Streptavidine-HRP is a conjugated protein consisting of the streptavidin protein and the enzyme horseradish peroxidase (HRP). Streptavidin has a high affinity for the vitamin biotin, while HRP is an enzyme that can catalyze a colorimetric reaction. The combination of these two components makes Streptavidine-HRP a useful tool in various biotechnological and immunological applications.
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2 protocols using streptavidine hrp
1
Fabrication of Microcrystalline Wax Immunoassay
2
Photolysis-Mediated Caspase Inhibition Assay
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Intrinsic apoptosis was induced in HEK293T lysates for 1 h. Indicated inhibitors (0.1 or 1 mM) or DMSO (control) were incubated with the apoptotic lysates for 30 min at 37 C. For the samples undergoing photolysis, the inhibitors were irradiated for the first 5 mins of the incubation under a 365nm longwave UVA lamp (100 W). Then, residual caspase activity was measured by incubation with biotin-DEVD-AOMK (7, 1 mM) for 30 min at 37 C. Subsequently, samples were boiled with 4X sample buffer for 3 min at 95 C, separated by SDS-PAGE and transferred onto a nitrocellulose membrane, followed by blocking for 1 h at room temperature (3% milk powder in PBST). The membrane was incubated with streptavidine-HRP (Sigma-Aldrich; 1 mg/mL) at a 1:3500 dilution in PBST for 1 h at room temperature. The blot was then washed with PBST buffer 3 times for 10 min. The membrane was incubated for 2 min with freshly prepared ECL solution 1 (4.5 mL water, 0.5 mL Tris 1M, 22.5 mL of 90 mM coumaric acid DMSO stock, 50 mL of 250 mM luminol DMSO stock) and solution 2 (4.5 mL water, 0.5 mL Tris 1M, 3 mL hydrogen peroxide 30%) and visualized.
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