The infected cells were lyzed in lysis buffer containing 20 mM Tris, 100 mM NaCl, and 1% NP40. The lysates were separated on 7%, 10%, and 15% SDS-PAGE gels. Proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences). The nonspecific binding sites on the membrane were blocked with 5% blocking solution (Roche Diagnostics) for 1 h before proteins were allowed to react with specific primary antibodies against TLR3 (Thermo Fisher Scientific), TLR7 (R&D Systems), iNOS (Santa Cruz Biotechnology), TLR9 (Santa Cruz Biotechnology), caspase-11 (Abcam), GSDMD (Abcam), and Actin (Merck Millipore) at 4°C overnight. The membranes were washed three times with 0.1% PBS with Tween 20 (PBST) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (R&D Systems) for 1 h at room temperature. Thereafter, the membranes were washed four times with 0.1% PBST before a chemiluminescence substrate (Roche Diagnostics) was added and protein brands were detected by enhanced chemiluminescence.
Chemiluminescence substrate
Manufactured by Roche
Sourced in United States
Chemiluminescence substrate is a reagent used in laboratory assays to detect and quantify target analytes. It produces a luminescent signal when it reacts with an enzyme label, allowing for sensitive and specific detection of the analyte of interest. The core function of the chemiluminescence substrate is to provide the necessary components for this luminescent reaction to occur, enabling researchers to measure and analyze the presence and levels of their target molecules.
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Lab products found in correlation
2 protocols using chemiluminescence substrate
1
Western Blot Analysis of Immune Signaling
2
Proteomic Profiling of Dystrophin Complex
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For the gel-and liquid chromatography-based proteomic and biochemical profiling of the dystrophin complex and membrane fractions from skeletal muscles, analytical grade reagents and materials were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK), Sigma Chemical Company (Dorset, UK), Bio-Rad Laboratories (Hemel-Hempstead, Hertfordshire, UK) and National Diagnostics (Atlanta, GA, USA). Proteolytic digestion was carried out with sequencing grade modified trypsin and Lys-C enzymes from Promega (Madison, WI, USA). Materials for immunoblotting included Whatman nitrocellulose transfer membranes from Invitrogen (Carlsbad, CA, USA), chemiluminescence substrate from Roche Diagnostics (Mannheim, Germany) and the reversible membrane stain MemCode from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies were purchased from Abcam, Cambridge, UK (ab124798 to desmoglein isoform DSG1, ab2413 to fibronectin, ab58562 to biglycan, ab52488 to lactate dehydrogenase, ab14739 to the voltage-dependent anion-selective channel protein VDAC-1, and ab16048 to lamin-B1), and Sigma-Aldrich, Dorset, UK (L9393 to laminin). Peroxidase-conjugated secondary antibodies were obtained from Chemicon International (Temecula, CA, USA).
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