Cells were lysed and protein extracts boiled and loaded on 10% or 15% polyacrylamide gels. After electrophoretic separation, the proteins were transferred to PVDF membranes, which were blocked with 5% milk powder in TBS + 0.1% Tween 20 (TBS-T) for 1 hour. Incubation of the primary antibody in TBS-T was performed at 4°C overnight. Membranes were then washed and stained with secondary antibody. Chemiluminescence was elicited using Western Lightning Ultra from PerkinElmer or Clarity Western ECL Substrate from Bio-Rad Laboratories (Munich, Germany), respectively, according to the manufacturers' instructions. The following primary antibodies were used: anti‐Caspase 3, anti‐cleaved Caspase 3 (Asp175), anti‐Caspase 8 (1C12), anti-PARP, anti-pCHK1 (Ser345) (133D3), and anti-PRIM1 (8G10) from Cell Signaling Technology (Cambridge, UK); anti-ATR (N-19), anti‐Caspase 9 (H-170), anti-Cdc25A (5H51), anti-CHK1 (G4), anti‐Cyclin A (H-432), and anti-Wee1 (B-11) from Santa Cruz Biotechnology (Dallas, TX); and anti‐β-Actin (AC-15) from Sigma-Aldrich. HRP-conjugated anti-goat, anti-rat, anti-mouse, and anti-rabbit antibodies from Santa Cruz Biotechnology were used as secondary antibodies.
Anti pchk1 ser345 133d3
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Anti-pCHK1 (Ser345) (133D3) is a monoclonal antibody that recognizes the phosphorylated form of CHK1 protein at serine 345. This antibody is designed for research use and can be used to detect and study the activated form of CHK1, a key regulator of the DNA damage response pathway.
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2 protocols using anti pchk1 ser345 133d3
1
Immunoblotting of Apoptosis Markers
2
Immunoblotting Procedure for Protein Analysis
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Immunoblotting was performed as described previously39 (link). In short, cells were lysed and protein extracts were boiled and loaded on 10% polyacrylamide gels. After electrophoretic separation, the proteins were transferred to PVDF membranes, which were blocked with 5% milk powder in Tris-buffered saline + 0.1% Tween 20 (TBS-T) for 1 h. Incubation of primary antibody in TBS-T was performed at 4 °C overnight. Membranes were then washed and stained with secondary antibody. Chemiluminescence was elicited using Western Lightning Ultra from PerkinElmer (Waltham, MA, USA) or Clarity Western ECL Substrate from Bio-Rad Laboratories (Hercules, CA, USA), respectively, according to the manufacturers’ instructions. The following primary antibodies were used: anti-caspase 3, anti-cleaved caspase 3 (Asp175), anti-PARP, and anti-pCHK1(Ser345) (133D3) from Cell Signaling (Cambridge, UK), anti-CHK1 (G-4) and anti-POLD1 (A9) from Santa Cruz Biotechnology (Dallas, TX, USA), and peroxidase-conjugated anti-β-Actin (AC-15) from Sigma-Aldrich (Hamburg, Germany). HRP-conjugated anti-rabbit, anti-goat and anti-mouse antibodies from Santa Cruz Biotechnology were used as secondary antibodies.
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