The cells were lysed with Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, lL) containing 1 × phosphatase inhibitor cocktails 2, 3 (Sigma). For immunoprecipitation, 500 µg of protein precleared with Mammalian Protein Extraction Reagent was incubated with antibodies, as indicated, overnight and then with protein G-agarose (Thermo Fisher Scientific) for two hours. Cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes. The blot was probed with primary antibodies. The primary antibodies used for western bolt were SIRT6 (Cell Signaling Technology), cleaved PARP1 (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), BCL2 (Santa Cruz Biotechnology), BAX (Santa Cruz Biotechnology), Chk2 (Thr68) (Cell Signaling Technology), phosphorylated Chk2 (p-Chk2, Cell Signaling Technology), ATM (Cell Signaling Technology), phosphorylated ATM (Ser1981) (p-ATM, Cell Signaling Technology), P53 (Santa Cruz Biotechnology), phosphorylated P53 (Ser15) (p-P53,Santa Cruz Biotechnology), H2AX (Cell Signaling Technology), γH2AX (Cell Signaling Technology), IgG (Cell Signaling Technology), and GAPDH (Santa Cruz Biotechnology). The bands of western blot were quantified using an ImageJ software (https://imagej.nih.gov/ij ).
Chk2 thr68
Manufactured by Cell Signaling Technology
Sourced in United States
Chk2 (Thr68) is a rabbit monoclonal antibody that specifically recognizes the phosphorylated form of the Chk2 protein at threonine 68. Chk2 is a serine/threonine-protein kinase that plays a crucial role in the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Protein g agarose, by Thermo Fisher Scientific (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Sirt6, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by CWBIO (1 mentions)
Eecl western blot kit, by CWBIO (1 mentions)
2 protocols using chk2 thr68
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were rinsed with PBS and scraped into radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitors (CW Biotech). Equal amounts (20-50 µg) of protein were separated on sodium dodecyl sulfate (SDS)polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Proteins were immunodetected using specific antibodies against β-actin (1:3000; CW Biotech; 0096), p16 (1:2000; Abcam; ab32034; Cambridge, MA, USA), p21 (1:100; Abclonal; AP0327; Wuhan, China), p27 (1:1000; Abcam; ab108349), p53 (1:50; Thermo Fisher Scientific; RM-9105; Fremont, CA, USA), EZH2 (1:500; Active Motif; 39902; Carlsbad, CA, USA), and DNA damage antibody including phospho-ataxia-telangiectasia mutated (ATM) (Ser1981), H2AX (Ser139), Chk1 (Ser345), and Chk2 (Thr68) (1:1000; Cell Signaling; 9947) overnight at 4°C. After washing with Tris buffered saline with Tween 20 (TBST) buffer, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Immunoblots were detected using an eECL Western Blot Kit (CW Biotech) following the manufacturer's instruction.
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