Western blotting of TLR2 and TLR4 in ESCs was performed according to the protocol published recently with some modifications (25 (link)). Blots were incubated for 2 hr with agitation in goat anti-TLR4 antibody (R&D, USA) at 1 μg/ml or 1:100 dilution of goat anti-TLR2 (Santa Cruz, USA) followed by 45 min incubation in HRP-conjugated rabbit anti-goat Ig (Sinabiotech, Iran). Bands were detected using an ECL Western blotting substrate kit and digital images were obtained with a Gel Logic 2200 imaging system (Kodak, Tokyo, Japan). Membranes were later stripped using Western Re-Probe (Calbiochem, SanDiego, CA, USA), re-incubated for 2 hr with agitation in polyclonal rabbit anti-beta actin antibody (Sigma) as loading control followed by 45 min incubation in HRP-conjugated sheep anti-rabbit Ig (Sinabiotech, Iran) and processed as above. In negative control blots, primary antibodies were substituted by equivalent dilutions of species-matched normal sera. Band densities of PCR and Western blot experiments were quantified as described elsewhere (25 (link)).
Goat anti tlr2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-TLR2 is a laboratory reagent that recognizes the toll-like receptor 2 (TLR2) protein. TLR2 is a pattern recognition receptor involved in the innate immune response. The antibody can be used to detect and study the expression and distribution of TLR2 in various cell and tissue samples.
Automatically generated - may contain errors
2 protocols using goat anti tlr2
1
Western Blotting of TLR2 and TLR4 in ESCs
2
Western Blot Analysis of Immune Markers
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Supernatants obtained after homogenization of cell pellets in lysis buffer of protein concentration 1.5 mg/ml were separated on the 5-20 % SDS-PAGE under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes blocked with Tris-buffered saline with 0.1 % Tween 20 (TBS) (Golias et al. 2013) . Blocked membranes were incubated with a primary antibody (Ab) diluted in TBS containing 0.1 % defatted milk (Promil, Nový Bydžov, Czech Republic) for 1 h at room temperature. Primary Abs: goat anti-IL-18 diluted 1:5,000, goat anti-TLR-2 and goat anti-TLR-4, both diluted 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Control recombinant rat IL-18 protein (R&D Systems, Minneapolis, MN, USA); rabbit anti-caspase-1 (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1,000. The membranes were incubated with peroxidase-conjugated secondary Ab: donkey anti-goat IgG, goat anti-rabbit IgG (Cell Signaling Technology) diluted 1:5,000 with TBS containing 1 % milk. Signal was detected using the West Femto Maximum Sensitivity Substrate kit (Pierce, Rockford, IL, USA) and exposed onto films (Santa Cruz Biotechnology).
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