Western blotting experiment was described previously (Zhao et al., 2019 (link)), and the detection of RIPK3 or MLKL oligomers was performed according to the method described by Wang et al. (2019) (link). The following antibodies were used for the experiments: anti-FADD (1:1000, 610399) and anti-RIPK1 (1:3000, 610458) (BD Transduction Laboratories, San Jose, CA); anti-phospho-RIPK3 (1:1000, ab195117), anti-MLKL (1:1000, ab172868), anti-phospho-MLKL (1:1000, ab196436) (Abcam, Cambridge, MA, United States); anti-RIPK1 (1:3000, 3493), anti-RIPK3 (1:3000, 15828), anti-Myc tag (1:2000, 2276), anti-HA tag (1:2000, 3724), anti-Flag tag (1:2000, 14793), anti-TRAF2 (1:1000, 4724), anti-phospho-RIPK1 (1:1500, 31122) and anti-phospho-RIPK3 (1:1000, 57220) [Cell Signaling Technology (CST), Beverly, MA]; anti-TRADD (1:1000, ABP52634) and anti-GAPDH (1:1500, ABP52783) (Abbkine, Redlands, CA, United States); anti-β-actin (1:3000, A5441) (Sigma-Aldrich). All western blot assays were performed three times, and the representative results are shown.
Anti ripk1
Manufactured by Abcam
Sourced in United States
Anti-RIPK1 is a lab equipment product that targets the RIPK1 protein. RIPK1 is a key regulator of cell death pathways. This product can be used to investigate the role of RIPK1 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti mlkl, by Abcam (4 mentions)
Anti ripk3, by Abcam (3 mentions)
Anti camkiiδ, by GeneTex (2 mentions)
Ab195117, by Abcam (1 mentions)
Ab196436, by Abcam (1 mentions)
Ab172868, by Abcam (1 mentions)
Anti ripk3, by Cell Signaling Technology (1 mentions)
Anti flag tag, by Cell Signaling Technology (1 mentions)
Anti myc tag, by Cell Signaling Technology (1 mentions)
Anti traf2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abbkine (1 mentions)
Anti ripk1, by BD (1 mentions)
Anti phospho ripk1, by Cell Signaling Technology (1 mentions)
Abp52634, by Abbkine (1 mentions)
Anti phospho ripk3, by Cell Signaling Technology (1 mentions)
Anti phospho mlkl, by Abcam (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti caspase 8, by Proteintech (1 mentions)
Cy3 labeled secondary antibody, by Beyotime (1 mentions)
5 protocols using anti ripk1
1
Detecting RIPK3 and MLKL Oligomers
2
Immunohistochemical Analysis of Necroptosis Signaling
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The gingival tissue samples were soaked in 4% paraformaldehyde for 24 hours, embedded in wax and cut to 3 μm in thickness. After regular deparaffinization, rehydration and antigen retrieval with heated citrate buffer (pH 6.0), the sections were incubated with anti-MLKL, anti-MLKL (phospho S358), anti-RIPK3, anti-RIPK1 (Abcam, US) or anti-caspase8 (Proteintech, China) overnight at 4 °C. Then, the slides were washed three times with phosphate-buffered saline and incubated with secondary antibodies (MaxVision, China) at room temperature for 30 min. Diaminobenzidine (DAKO, USA) was used as a chromogenic agent to detect antibody binding.
3
Quantitative Immunofluorescence Analysis
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Cells on coverslips were blocked (1 × PBS, 2% normal goat serum, and 0.1% Triton X-100) for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-RIPK1 (1:400; Abcam, Burlingame, CA, USA), anti-RIPK3 (1:400, Abcam), anti-MLKL (1:400, Abcam), and anti-CaMKIIδ (1:100; GeneTex, Irvine, CA, USA). Cells were washed three times with 0.1 M PBS and then incubated with Cy3-labeled secondary antibody (1:400; Beyotime, Shanghai, China) for 1 h at room temperature. The cells were then photographed under a fluorescent microscope (Leica, Microsystems, Wetzlar, Germany) with an excitation wavelength of 550 nm and an emission wavelength of 570 nm.
4
Molecular Mechanisms of Pentobarbital-Induced Necroptosis
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Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).
5
Protein Extraction and Western Blot Analysis
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Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitors. Homogenates were maintained in ice for 30 min and centrifuged at 15 000 × g for 10 min at 4 °C, and the supernatant was recovered. Protein concentration was determined by BCA protein assay kit (Life, New York, NY, USA). Proteins were resolved in SDS-PAGE (10% polyacrylamide), transferred to PVDF membrane, and incubated with primary antibodies. The reactions were followed by incubation with peroxidase labeled secondary antibodies (Life). Primary antibodies used were: anti-RIPK1 (1:800, Abcam), anti-RIPK3 (1:800; Abcam), anti-MLKL (1:1000, Abcam), anti-β-actin (1:5000, Abcam), anti-Na+-K+-ATPase (1:400; Santa Cruz, Santa Cruz, CA, USA), anti-CaMKIIδ (1:800, GeneTex), anti-p-CaMKII (1:800, Thermo), and anti-ox-CaMKII (1:600; Millipore, Bedford, MA, USA). CaMKII activation was assessed by measuring phosphorylation and oxidation levels.
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