NF-κB Pathway Sampler Kit, Akt, p-Akt (Ser 308), p-Akt (Thr 473), p21, p-CDK2, CDK2, cyclinA, cyclinD1, mTOR Pathway Sampler Kit, and Apoptosis sampler Kit were obtained from Cell Signaling Technology (Beverly, MA, USA). All the cell lines were treated with NVP-Bez235 and lenalidomide in single way or combination for 72 h. Bortezomib was purchased from Selleck (Huston, USA). Cells were performed with 5 nM Bortezomibe for 24 and 48 h, respectively. Then the samples in each group were collected and separated by standard SDS-PAGE gel electrophoresis and transferred to NC membrane. Blocked with 5 % non-fat milk supplemented in 0.1 % TBST, membrane was probed with primary antibodies at 4 °C overnight. After washed with TBST, membrane was incubated with HRP-conjugated secondary antibodies (Santa Cruz, CA) for one hour. The bands were detected and quantified in Image Lab software (Bio-Rad Laboratories, California, USA).
Apoptosis sampler kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Apoptosis Sampler Kit is a collection of primary antibodies used to detect key proteins involved in the apoptosis pathway. The kit includes antibodies targeting Caspase-3, Caspase-8, Caspase-9, PARP, and Bcl-2 family members. These antibodies can be used to analyze the activation of apoptosis-related proteins in a variety of cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Extraction buffer, by iNtRON Biotechnology (2 mentions)
Cyclin a, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
P cdk2, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nf κb pathway sampler kit, by Cell Signaling Technology (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti β actin ab, by Merck Group (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti bak, by Cell Signaling Technology (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
Confocal microscope, by Leica (1 mentions)
5 protocols using apoptosis sampler kit
1
Proteomic Analysis of NVP-Bez235 and Lenalidomide Effects
2
Western Blot Analysis of Gastric Cancer
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Tumor tissue specimens were harvested from EBV(+) and EBV(−) human gastric carcinoma SNU719 or MKN74 bearing mice and lysed using buffer solution (PRO-PREP Protein Extraction Solution, Intron, Korea). Protein level in tumor tissue lysates was measured using the Bradford Assay Plus Kit (Gen DEPOT. Inc., Baker, TX, USA), separated by electrophoresis, and transferred to nitrocellulose membranes. Membranes were incubated with first and second antibodies and blots were detected using enhanced chemiluminescent (ECL) reagents (GE Healthcare Life Sciences, Logan, UT, USA). The primary antibodies used were anti-p53 (clone BP53-12, Millipore, Billerica, MA, USA), anti-p21, anti-Bax (Cell signaling, Danvers, MA, USA), Apoptosis Sampler Kit (#9915, Cell signaling), and anti-β-actin Ab (Sigma Aldrich) [39 (link),40 (link)].
3
Quantification of Apoptosis Proteins in Tumor Tissue
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The levels of apoptosis-related proteins in tumor tissue were determined by Western blot analysis [18 (link)]. The specimens were lysed with an extraction buffer (Intron Biotechnology, Seoul, Korea) to extract proteins, and protein concentration was determined by Coomassie (Bradford) Protein Assay (genDEPOT, Katy, TX, USA). The proteins were separated by electrophoresis and transferred onto nitrocellulose membranes (0.45-µM pore size, Merck Millipore, Burlington, MA, USA). The membranes were blotted with the following primary and secondary antibodies: Apoptosis Sampler Kit (#9915), anti-caspase-8 Ab (#9746), anti-GAPDH (#5174), anti-rabbit IgG (#7074), and anti-mouse IgG (#7076), all obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Proteins were visualized using chemiluminescence detection (FluorChem E system, ProteinSimple, San Jose, CA, USA) and quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). The expression levels relative to GAPDH were determined.
4
Apoptosis Protein Expression Analysis
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Apoptosis-related protein expression in tumor tissues was examined using Western blotting analysis [50 (link)]. The specimens were lysed using extraction buffer (Intron Biotechnology, Seoul, Korea) to extract proteins, which were quantified by Bradford (Coomassie) Protein Assay (GenDEPOT, Katy, TX, USA). Proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVFD) microporous membranes (Merck Millipore, Burlington, MA, USA). Membranes were incubated with the primary and secondary antibodies. Anti-Akt (#9272), anti-p-Akt (#4060), anti-p53 (#2527), anti-Bax (#2774), anti-Bak (#21205), apoptosis Sampler Kit (#9915), anti-GAPDH (#5174), anti-rabbit IgG (#7074), and anti-mouse IgG (#7076) were obtained from Cell Signaling Technology (CST; Danvers, MA, USA) and used for the incubation. Proteins were visualized using chemiluminescence detection (ECL System, Bio-Rad, Hercules, CA, USA) and quantified using the Image J program (National Institutes of Health, Bethesda, MD, USA).
5
Apoptosis Induction by Carmustine and Curcumin
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1x104 U251 cells were seeded in 8-well chamber slides. Cells were treated with carmustine (150 µM), curcumin (20µM) and combination of both (150µM+20µM). An equivalent amount of ethanol required to make the concentration of drug was added in the control well. After 48 h incubation, media with drug was removed and washed thrice with PBS and fixed with ice-cold 4% paraformaldehyde at 4°C for 15 min. After washing with PBS, post fixing cells were blocked using 10% normal goat serum plus Triton X for 45 min at room temperature. Cells were incubated with Cleaved Caspase 3 primary antibody (Apoptosis sampler kit Cell signaling technologies #9664) overnight at 4°C. Cells were washed well with PBS and incubated with anti-rabbit secondary antibody tagged with Alexa fluor 488 (Abcam 150077) for 1 h. At the end of incubation, nucleus was stained with Hoechst 33342 for 5 min. Finally, the cells were washed 3 times with PBS and imaged with Leica confocal microscope.
Apoptotic Index was calculated by the formula:
Apoptotic Index = (Cleaved caspase 3 positive cells/Total number of cells) x 100.
Apoptotic Index was calculated by the formula:
Apoptotic Index = (Cleaved caspase 3 positive cells/Total number of cells) x 100.
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