Western Blot analysis was performed as described before35 (link). In brief, cells were homogenized in RIPA lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). Protein lysates were resolved on SDS-PAGE gels before being transferred to the PVDF membrane. Anti-FADD, anti-Caspase-8, anti-RIP, and anti-RIP3 antibodies were purchased from Santa Cruz (sc-271748, sc-56070, sc-133102, and sc-374639 respectively). Mouse monoclonal anti-Actin antibody was used as a loading control. The densities of bands were quantitated using ImageJ software.
Sc 133102
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-133102 is a lab equipment product manufactured by Santa Cruz Biotechnology. It is designed for general laboratory use. The core function of Sc-133102 is to assist researchers in their experimental procedures. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Sc 374639, by Santa Cruz Biotechnology (2 mentions)
Ab195117, by Abcam (2 mentions)
Ab196436, by Abcam (2 mentions)
Ab4051, by Abcam (2 mentions)
Ab25901, by Abcam (2 mentions)
Ab172868, by Abcam (2 mentions)
Ab52298, by Abcam (2 mentions)
Chemidoc xrs image system, by Bio-Rad (2 mentions)
Sc 32233, by Santa Cruz Biotechnology (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Sc 56070, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 8, by Santa Cruz Biotechnology (1 mentions)
Sc 271748, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab179434, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
5 protocols using sc 133102
1
Apoptotic Signaling Pathway Analysis
2
Co-immunoprecipitation of RIP1, K63, and GAPDH
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Co-immunoprecipitation assay (CO-IP) was performed as previously described [25] (link). Brie y, 300-500 µl tissue lysate was incubated with 0.5-2 µg antibody against RIP1 (sc-133102; Santa Cruz; 1:1000), K63 (ab179434; Abcam; 1:1000), or GAPDH (ab181602; Abcam; 1:1000) for 3 h at 4°C. 50-µlProtein G-agarose beads (Beyotime) was then added and incubated overnight. After washing, samples were boiled for 10 min in sample-loading buffer, then subjected to SDS-PAGE (5%-15%) and analyzed by Western blot.
3
Immunoprecipitation and Western Blotting
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The cells were collected as previously described in the western blotting section. Cells were lysed in binding buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor). The monoclonal antibody against RIPK1(CA#sc-133102, mouse, Santa Cruz, CA, United States) or against RIPK3 (CA# sc-374639, mouse, Santa Cruz, CA, United States) or a matched-isotype antibody IgG (CST) was incubated with Protein A/G Magnetic Beads (MCE, Monmouth Junction, NJ, United States) at 4°C overnight. On the second day, a total of 1 mg protein sample lysate was incubated with an antibody-beads complex for more than 8 h. Then, the magnetic rack was used to precipitated the antibody-beads complexes. Then, the complexes were washed for three times and then eluted from beads by boiling at 95°C with 4 × LDS loading buffer (CA#161-0747, Biorad, United States). Finally, these protein samples were resolved by SDS/PAGE assay and immunoblotted with antibodies as indicated.
4
Immunoblot Analysis of Cell Stress Signaling
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Following ELF-EMF exposure, cells were collected and homogenized using lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Triton-X100 and proteases inhibitor cocktail). Achieved cell lysates were than subjected to centrifugation at 15,000 g for 30 min at 4 °C and supernatant was collected for further analysis. Proteins in supernatant were then separated by SDS-PAGE and transferred on a PVDF membrane for further visualization. Using a 5% bovine serum albumin, blotted PVDF membranes were blocked for 1 h and then probed using primary antibodies against caspase-3 (ab4051, Abcam), caspase-8 (ab25901, Abcam), caspase-9 (ab52298, Abcam), RIPK1 (sc-133102, Santa Cruz Biotechnology), phosphorylated RIPK1 (p-RIPK1) (#44590, CST), RIPK3 (#13526, CST), phosphorylated RIPK3 (p-RIPK3) (ab195117, Abcam, Cambridge, MA, United States), MLKL (ab172868, Abcam), phosphorylated MLKL (p-MLKL) (ab196436, Abcam) and GAPDH (sc-32233, Santa Cruz Biotechnology). Following addition of secondary antibodies and washing with TBST for 3 times, the immunoreactive bands were visualized utilizing a ChemiDoc XRS Image System (Bio-Rad Laboratories, Hercules, CA, USA). Obtained images were then quanti ed ImageJ analysis software.
5
Immunoblot Analysis of Cell Stress Signaling
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Following ELF-EMF exposure, cells were collected and homogenized using lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Triton-X100 and proteases inhibitor cocktail). Achieved cell lysates were than subjected to centrifugation at 15,000 g for 30 min at 4 °C and supernatant was collected for further analysis. Proteins in supernatant were then separated by SDS-PAGE and transferred on a PVDF membrane for further visualization. Using a 5% bovine serum albumin, blotted PVDF membranes were blocked for 1 h and then probed using primary antibodies against caspase-3 (ab4051, Abcam), caspase-8 (ab25901, Abcam), caspase-9 (ab52298, Abcam), RIPK1 (sc-133102, Santa Cruz Biotechnology), phosphorylated RIPK1 (p-RIPK1) (#44590, CST), RIPK3 (#13526, CST), phosphorylated RIPK3 (p-RIPK3) (ab195117, Abcam, Cambridge, MA, United States), MLKL (ab172868, Abcam), phosphorylated MLKL (p-MLKL) (ab196436, Abcam) and GAPDH (sc-32233, Santa Cruz Biotechnology). Following addition of secondary antibodies and washing with TBST for 3 times, the immunoreactive bands were visualized utilizing a ChemiDoc XRS Image System (Bio-Rad Laboratories, Hercules, CA, USA). Obtained images were then quanti ed ImageJ analysis software.
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