Esculin (≥98%, HPLC) was purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Protein G-Sepharose™ 4B beads, LPS, fMLP and calcein-AM was obtained from Sigma-Aldrich (St. Louis, MO). Recombinant human intercellular adhesion molecules 1 (rhICAM-1) or human IgG were purchased from R&D Systems (Abingdon, VA). ELISA kits for TNF-α, IL-6 and IL-1β were obtained from Invitrogen (Vienna, Austria). ELISA kit for MCP-1 was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against IκBα (ab76429), p-IκBα (ab133462), Vav1 (ab97574) and phospho-Vav1 (ab76225) were acquired from Abcam (Cambridge, MA). Anti-PAK1 (#2602), anti-phospho-PAK1 (#2601), anti-LIMK1 (#3842), anti-phospho-LIMK1(#3841), anti-cofilin (#3318), anti-phospho-cofilin (#3311), anti-p65 (#8242), anti-p-p65 (#3033), anti-GST (#2624), anti-Rac1 (#4651), anti-ICAM-1 (#4915), anti-β2 integrin (#73663) and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Berkeley, CA). Enhanced Supersignal West Pico Chemiluminescence (ECL) substrate kit was purchased from Thermo Scientific (Rockford, IL).
Protein g sepharose 4b beads
Manufactured by Merck Group
Sourced in United States
Protein G-Sepharose™ 4B beads are a chromatographic resin composed of crosslinked agarose beads with covalently coupled Protein G. Protein G is a bacterial protein that binds to the Fc region of immunoglobulins. The beads can be used for the purification and isolation of antibodies and immunoglobulins from various sample sources.
Automatically generated - may contain errors
Lab products found in correlation
Ab76429, by Abcam (1 mentions)
Ab97574, by Abcam (1 mentions)
Rhicam 1, by R&D Systems (1 mentions)
Ab76225, by Abcam (1 mentions)
Calcein am, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ab133462, by Abcam (1 mentions)
3 protocols using protein g sepharose 4b beads
1
Esculin Inhibits Inflammatory Responses
2
Cell–Cell Contact Immunoprecipitation
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Cells were induced to form cell–cell contacts for 30 min and then lysed in lysis buffer (1% Triton X-100, 15% Glycerol, 50 mM Tris–HCl pH 8, 150 mM NaCl, and 5 mM EDTA, 5 μg/ml leupeptin, 5 μg/ml pefabloc, 5 μg/ml, pepstatin, 50 mM phenylmethylsulfonyl fluoride, 20 mM sodium fluoride, 1 mM sodium pyruvate, 1 mM sodium orthovanadate and 20 mM β-glycero-phosphate). After centrifugation for 5 min, pre-cleared supernatants were immunoprecipitated for 1 h with 5 μl of the indicated antibody bound to 40 μl slur of protein A or protein G-Sepharose-4B beads (Sigma). Beads were washed three times in lysis buffer and SDS–PAGE sample buffer added to beads.
3
Chromatin Immunoprecipitation of Neural Stem Cells
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Neural stem and progenitor cell pellets and dentate gyri isolated from brain slices containing hippocampus were homogenized in lysis buffer containing 1% SDS, 50 mM Tris-HCl (pH 8.0), and 10 mM EDTA and sonicated on ice with 6 10-s pulses with a 20-s interpulse interval. Sample debris was removed via centrifugation, and supernatants were immunoprocessed with 2 µg of anti-FoxO1, anti-FoxO3a, anti-histone H3 lysine 9 acetylation (H3K9ac), or control IgG overnight at 4 °C. Immunocomplexes were collected via incubation with protein-G Sepharose 4B beads (Sigma-Aldrich, St. Louis, MO, USA) for 2 hours at 4 °C. After 7 sequential washes, immunocomplexes were eluted from beads by vortexing in elution buffer (1% SDS and NaHCO 3 0.1 M; pH 8.0). NaCl was added (final concentration 0.33 M), and cross-linking was reversed by incubation overnight at 65 °C. DNA fragments were purified using a PCR DNA fragments purification kit (Geneaid Biotech Ltd., New Taipei City, Taiwan). The antibodies and primer sequences are shown in Supplementary Tables S1 andS3, respectively.
PCR reaction was performed in triplicate. Data are expressed as percentage of input calculated by the "adjusted input value" method according to the manufacturer's instructions (ThermoFisher Scientific ChIP Analysis, Carlsbad, CA, USA).
PCR reaction was performed in triplicate. Data are expressed as percentage of input calculated by the "adjusted input value" method according to the manufacturer's instructions (ThermoFisher Scientific ChIP Analysis, Carlsbad, CA, USA).
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