Colorectal cancer specimens and colorectal cancer cells were lysed with RIPA buffer (TargetMol, USA) containing protease inhibitor and phosphatase inhibitor. The supernatant was collected after centrifugation at 4°C for 30 min. After the protein was quantified and denatured, the denatured protein was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking the membrane with 3% bovine serum albumin for 1 h, primary antibody was added and incubated overnight at 4°C. After removal of the primary antibody, the membrane was washed three times with TBST and incubated with secondary antibody at room temperature for 1 h. Immune complexes were detected by an enhanced chemiluminescence system (Life Tec, USA), and protein bands were analyzed and quantified using ImageJ software (version 11). The primary antibodies were as follows: caspase 3 (Abclonal, China), caspase 9 (Abclonal, China), cleaved caspase 3 (CST, USA), CYP24A1 (CST, America), cleaved caspase 9 (Abclonal, China), γh2A (Abclonal, China), Ki67 (Abclonal, China) and GAPDH (Abclonal, China). The secondary antibody was purchased from Abclonal (China).
Cleaved caspase 9
Manufactured by ABclonal
Sourced in China
Cleaved caspase 9 is a laboratory reagent used in research applications. It is an enzyme fragment generated by the proteolytic cleavage of the full-length caspase 9 protein. Caspase 9 is a key mediator of apoptosis, or programmed cell death, and the cleaved form can be used to study this cellular process.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by ABclonal (2 mentions)
Caspase 9, by ABclonal (2 mentions)
Cleaved parp1, by ABclonal (2 mentions)
Cleaved caspase 3, by ABclonal (2 mentions)
Bcl2 associated x (bax), by ABclonal (2 mentions)
Bcl 2, by ABclonal (2 mentions)
Ripa buffer, by Targetmol (1 mentions)
Caspase 3, by ABclonal (1 mentions)
Auranofin, by Merck Group (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Dimethyl sulfoxide (dmso), by MedChemExpress (1 mentions)
Ubiquitin, by ABclonal (1 mentions)
Lamina c, by ABclonal (1 mentions)
Parp1, by ABclonal (1 mentions)
Ha tag, by ABclonal (1 mentions)
Flag tag (flag), by ABclonal (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
β actin, by ABclonal (1 mentions)
Mg132, by MedChemExpress (1 mentions)
Col1a1, by ABclonal (1 mentions)
3 protocols using cleaved caspase 9
1
Colorectal Cancer Cell Protein Analysis
2
Synthesis and Application of LW-216 Compound
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LW-216 (C30H31NO5, MW: 485.57, purity>96 %, CAS number: 2146090-18-6) was synthesized by Prof. Zhiyu Li in China Pharmaceutical University [22 (link)]. Auranofin (C20H34AuO9, MW: 678.48, purity>99 %, CAS number: 34031-32-8) was obtained from Yuancheng Gongchuang Technology (Wuhan, China). Both LW-216 (10 mM) and Auranofin (1 mM) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St.Louis, MO, USA) as a stock solution at -20 °C and diluted with medium before in vitro experiments. MG-132 (10 mM) and Cycloheximide (CHX, 10 mg/mL) were purchased from MedChemExpress (NJ, USA), which were both dissolved with DMSO. The final concentration of DMSO did not exceed 0.1 %. Primary antibodies for TrxR1, TrxR2, Ubiquitin, HA-Tag, Flag-Tag, Bax, Bcl-2, PARP1, Cleaved PARP1, Caspase-9, Cleaved Caspase-9, AIF, CytC, VDAC, Lamin A/C, Ki67, Cleaved Caspase-3, COL1A1 and β-actin were obtained from ABclonal Technology (Wuhan, China).
3
Western Blot Analysis of Apoptosis-Related Proteins
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Tissues were homogenized in RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology) and incubated on ice for 30 min. After centrifugation at 12 000 rpm for 15 min, the supernatant was used for western blotting. Western blotting was performed as previously described8 (link). The blots were incubated overnight with primary antibodies against the following target proteins: Bax (1:1000, ABclonal, China), Bcl-2 (1:1000, ABclonal, China), cleaved PARP-1(1:2000, ABclonal, China), cleaved caspase-3 (1:1000, ABclonal, China), cleaved caspase-9 (1:1000, ABclonal, China), P53 (1:1000, Cell Signalling Technology), ERK (1:1000, ABclonal, China), p-ERK (1:1000, ABclonal, China), MEK (1:1000, ABclonal, China), p-MEK (1:1000, ABclonal, China), and GAPDH (1:1000, ABclonal, China). Western blotting was performed using enhanced chemiluminescence reagent (EpiZyme) and visualized using an electrophoretic gel imaging system (Tanon).
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