For immunoprecipitation (IP), whole-cell extracts were lysed in an IP buffer composed of 1% (vol/vol) NP-40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5) and the complete protease inhibitor cocktail (Roche). Cell lysates were collected and incubated with 1 μg of the corresponding antibodies together with protein G Plus-Agarose Immunoprecipitation reagent (Santa Cruz). After overnight incubation at 4 °C, agaroses were washed five times and boiled with the IP buffer for SDS-PAGE. For immunoblotting analysis, cells were lysed with a lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 50 mM Tris-HCl, pH 7.5) supplemented with the protease inhibitor cocktail (Roche), and were incubated with the following antibodies: anti-FLAG (Proteintech, 20543-1-AP), anti-HA (Proteintech, 51064-2-AP), anti-His (Proteintech, 66005-1-lg), anti-RIP1 (Santa Cruz, sc-133102), anti-RIP3 (Santa Cruz, sc-374639), anti-RIP1(Santa Cruz, sc-133102), anti-Myc (Proteintech, 67447-1-Ig), anti-pRIP3 (Abcam, ab205421), anti-mouse pMLKL (Abcam, ab196436), anti-human pMLKL (Abcam, ab206336), anti-MLKL (Abcam, ab243142), anti-Actin (Abcam, ab8226), and anti-TRIM25 (Abcam, ab167154).
Anti rip1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-RIP1 is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to the RIP1 protein, enabling detection and analysis of this target in experimental settings. The core purpose of Anti-RIP1 is to provide researchers with a tool for investigating the RIP1 protein and its role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti rip3, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 8, by Santa Cruz Biotechnology (1 mentions)
Ab196436, by Abcam (1 mentions)
Ab243142, by Abcam (1 mentions)
Ab205421, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab167154, by Abcam (1 mentions)
Protein g plus agarose immunoprecipitation reagent, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti mcl 1, by Santa Cruz Biotechnology (1 mentions)
Anti hsp70, by Cell Signaling Technology (1 mentions)
Anti cdk4, by Proteintech (1 mentions)
Nec 1, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti parp, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
4 protocols using anti rip1
1
Immunoprecipitation and Immunoblotting Protocol
2
Western Blot Analysis of Key Necroptosis Regulators
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To evaluate the effect of CIS and AZM on RIP1, RIP3, MLKL, and caspase-8, 50 µg protein was subjected to SDS-PAGE followed by transfer onto PVDF membranes. Then, 5% BSA was used for blocking, and the membranes were then incubated with anti-RIP1 (sc-133102—Santa Cruz, Dallas, TX, USA), anti-RIP3 (sc-374639—Santa Cruz, Dallas, TX, USA), anti-MLKL (YPA2507—Biospes, Chongqing, China), anti-caspase-8 (sc-70501—Santa Cruz, USA), and anti-β-actin (sc-8432—Santa Cruz, Dallas, TX, USA) overnight at 4 °C. After washing in TBST, the membranes were incubated with the secondary antibodies for 1 h at RT, washed and visualized using the BCIP/NBT substrate detection kit (GeneMed Biotechnologies, San Francisco, CA, USA). The intensity of the developed bands was determined using ImageJ (NIH, Bethesda, MD, USA).
3
Apoptosis and Necroptosis Pathway Regulation
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DHQ3 and 17-DR were obtained as described previously. They were dissolved in dimethyl sulfoxide (DMSO, Biosharp, Hefei, China) and stored at −20 °C. MG132, Nec-1, DAPI (4,6-diamidino-2-phenylindole), and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PI assay kits were purchased from Beyotime Institute of Biotechnology (Wuhan, China) and the Annexin V FITC/PI apoptosis detection kit was purchased from Nanjin KeyGen Biotech (Nanjing, China). The ATP Assay kit was purchased from Merck KGaA (Darmstadt, Germany). Lipofectamine 2000 was purchased from Invitrogen (USA). The following antibodies were used: anti-Mcl-1, anti-PARP, anti-RIP1, anti-RIP3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Bcl-2, anti-Bax, anti-HIF1a, anti-CDK4, anti-Her2, anti-EGFR (Proteintech, Chicago, IL, USA); anti-Hsp70, anti-Hsp90, anti-Akt (Cell Signaling Technology, Beverly, Massachusetts, USA); anti-caspase 3 and anti-caspase 8 (ENZO, Switzerland); anti-C-Raf (Abcam, Cambridge, MA, USA); and anti-β-actin (BioSharp, Hefei, China).
4
Quantification of Oxidative Stress Markers
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Whole cell or tissue samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX) with protease inhibitor cocktail (Thermo Scientific) and 40 μg total protein was used for immunoblot analysis according to our previous protocols [27] . Blots were probed with anti-Klotho (1:200; Thermo Scientific), anti-3-nitrotyrosine (1:500; Abcam, Cambridge), anti-SOD2 (1:500; Santa Cruz Biotechnology), anti-RIP1 (1:100; Santa Cruz Biotechnology), anti-RIP3 (1:500; Santa Cruz Biotechnology), anti-IL-1 beta (1:1000; Abcam), anti-tubulin (1:1000; Abcam), anti-beta-actin (1:2000; Abcam), anti-catalase (1:1000; Abcam), anti-GPX4 (1:1000; Abcam), anti-t-FoxO1 (1:1000; Cell Signaling Technology, Beverly, CA), antip-FoxO1 (1:1000; Cell Signaling Technology), anti-t-FoxO3a (1:1000; Cell Signaling Technology), anti-p-FoxO3a (1:1000; Cell Signaling Technology), or anti-GAPDH (1:1000; Cell Signaling Technology) antibodies overnight at 4 °C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signaling Technology). After washing, specific signals were determined using an ECL kit (Thermo Scientific) on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai). Gray values were analyzed with ImageJ software.
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