HSF-OAs, as plated above, were lysed in 50 μL lysis buffer (Qproteome Mammalian Protein kit; Qiagen) according to manufacturer's instructions and centrifuged at 12000×g at 4 °C for 10 min to remove the cellular debris. Lysates from all time-points were prepared for western blot analysis by boiling in Bolt Reducing Buffer and Bolt LDS Sample Buffer (ThermoFisher Scientific, Waltham, MA). The lysates were separated by SDS-PAGE (8%) and subjected to western blot analysis using an anti-COX2 rabbit monoclonal primary antibody (1:1000, ab62331; Abcam, Cambridge, MA) and a goat anti-rabbit IgG secondary antibody (1:10000, Cat# 7074P2, Cell Signaling, Danvers, MA). The COX2 protein levels were normalized to α-tubulin after stripping and reprobing with Reblot Plus (Millipore, Billerica, MA) and a horseradish peroxidase-conjugated α-tubulin antibody (1:5000, DM1A, Cat# 12351S, Cell Signaling, Danvers, MA), respectively.
Qproteome mammalian protein kit
Manufactured by Qiagen
Sourced in Germany
The Qproteome Mammalian Protein kit is a product developed by Qiagen for the extraction and purification of proteins from mammalian cells and tissues. The kit provides a streamlined protocol for the isolation of total cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Re blot plus, by Merck Group (1 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved parp1, by Abcam (1 mentions)
Anti caspase 9, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl substrate, by Abcam (1 mentions)
Dc detergent compatible protein assay, by Bio-Rad (1 mentions)
Chemidoc machine, by Bio-Rad (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti cytochrome c, by Abcam (1 mentions)
2 protocols using qproteome mammalian protein kit
1
COX2 Protein Expression Analysis
2
Apoptosis Regulation in Leukemia Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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KG-1 cells were plated in a 6-well plate at a density of 106 cells/mL before treatment with two increasing concentrations of ASEE for 24 h (38 and 76 μg/mL). Control cells were treated with RPMI media. Total proteins were extracted using the Q-proteome Mammalian Protein kit (Qiagen, Hilden, Germany) and quantified using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE; transferred to PVDF (Polyvinylidene fluoride) membranes which were blocked with 5% skimmed milk; and then incubated with primary antibodies anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cytochrome-c, anti-cleaved PARP-1, anti-Bax, anti-Bcl2, and anti-caspase-9 (Abcam, Cambridge, UK), anti-p53, and anti-caspase-8 (Elabscience, Houston, TX, USA) at the manufacturer’s recommended concentrations. After washing and incubation with a secondary antibody (Bio-Rad, Hercules, CA, USA), membranes were washed and image development was done using the Clarity™ Western ECL Substrate (Abcam, Cambridge, UK) on the ChemiDoc machine (BioRad, Hercules, CA, USA). Blot bands were quantified using the ImageJ computer program to calculate the relative expression of proteins.
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