Rabbit anti phospho chk1 ser345

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phospho-Chk1 (Ser345) is an antibody that specifically recognizes the phosphorylated form of the Chk1 protein at serine 345. It is used to detect and measure the levels of activated Chk1 in cells.

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4 protocols using rabbit anti phospho chk1 ser345

Protein Detection Assay Workflow

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Equal number of cells were collected for each sample and lysed in Laemmli sample buffer supplemented with protease inhibitor cocktail (Sigma) and beta-mercaptoethanol (5%) by heating at 95°C for 5 min. Proteins were separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore). Primary antibodies were: rabbit anti-HLTF (Abcam ab183042), mouse anti-alpha-tubulin (Sigma T9026), rabbit anti-phospho-Chk1 (Ser345) (Cell Signaling Technology 2348), mouse anti-Chk1 (sc-8408, G4, Santa Cruz), rabbit anti-phospho-RPA32 (Ser33) (Bethyl Laboratories A300–246A), rabbit anti-phospho-RPA32 (Ser4/8) (Bethyl Laboratories A300–245A), rabbit anti-phospho-Histone H2A.X (ser139) (Cell Signaling Technology 9718), mouse anti-RPA34 (Millipore NA19L), mouse anti-PCNA (sc-56, Santa Cruz), rabbit anti-PRIMPOL (Mouron et al., 2013 (link)), rabbit anti-Histone H3 (Abcam ab1791), mouse anti-REV1 (sc-393022, Santa Cruz), and mouse anti-GAPDH (Abcam ab8245). Secondary antibodies were goat anti-rabbit HRP (Molecular Probes G21234) and goat anti-mouse HRP (Invitrogen 81–6520). Chemi-luminescence was carried out using the Immobilon HRP substrate (Millipore WBKLS0500), and blots were imaged with a FluorChem HD2 from Alpha Innotech.

Bai G., Kermi C., Stoy H., Schiltz C., Bacal J., Zaino A.M., Hadden M.K., Eichman B., Lopes M, & Cimprich K.A. (2020). HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis. Molecular cell, 78(6), 1237-1251.e7.

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Western Blotting and Cell Fractionation

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Western blotting was performed as already described^{55 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643), mouse anti-cyclin A2 (#4656), rabbit anti-BCL2 (#2876), rabbit anti-BAX (#2772), rabbit anti-cyclin B1 (#12231), rabbit anti-PARP-1 (#9532), rabbit anti-phospho-ATR (Ser428) (#2853), rabbit anti-phospho-CHK1 (Ser345) (#2348), rabbit anti-phospho-CDC2 (Tyr15) (#4539), rabbit anti-phospho-H2A.X (Ser139) (#9718), rabbit anti-phospho-Histone H3 (Ser10) (#3377), rabbit anti-phospho-WEE1 (Ser642) (#4910) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CDC2 (sc-954), mouse anti-Myc (sc-40), mouse anti-HSP90 (sc-13119) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-SMO (ST1718) (Merck Millipore, Burlington, MA, USA). Chemiluminescent detection was used. Cell fractionation was performed as previously described^{56 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643) (Cell Signaling Technology), goat anti-fibrillarin (D-14), and goat anti-GAPDH (V-18) (Santa Cruz Biotechnology).

Pietrobono S., Santini R., Gagliardi S., Dapporto F., Colecchia D., Chiariello M., Leone C., Valoti M., Manetti F., Petricci E., Taddei M, & Stecca B. (2018). Targeted inhibition of Hedgehog-GLI signaling by novel acylguanidine derivatives inhibits melanoma cell growth by inducing replication stress and mitotic catastrophe. Cell Death & Disease, 9(2), 142.

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Evaluation of Stress Responses in INS 832/13 Cells

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INS 832/13 cells were obtained from Dr Christopher Newgard (Duke University). MEF were purchased from American Type Culture Collection. Male Sprague–Dawley rats were purchased from Harlan. Connaught Medical Research Laboratories (CMRL) 1066 medium and β-mercaptoethanol were purchased from Thermo Fisher Scientific. RPMI1640 medium, Dulbecco's modified Eagle's medium, trypsin (0.05% in 0.53 mM EDTA), l-glutamine, sodium pyruvate, Hepes, and penicillin–streptomycin were purchased from Corning. Fetal bovine serum was purchased from HyClone. DPTA/NO was purchased from Cayman Chemical. 2-Deoxyglucose (2-DG), camptothecin, hydroxyurea, and rotenone were purchased from MilliporeSigma. Primary and secondary antibodies used for Western blot and immunofluorescence were purchased as follows: mouse anti-γH2AX (Ser139) from MilliporeSigma; rabbit anti-phospho-KAP1 (Ser824) and rabbit anti-glucokinase from Abcam; rabbit anti-phospho-Chk1 (Ser345) from Cell Signaling Technology; mouse anti-GAPDH from Thermo Fisher Scientific; guinea pig anti-insulin from DakoCytomation; horseradish peroxidase (HRP)–conjugated donkey anti-mouse, HRP-conjugated donkey anti-rabbit, and Cy3-conjugated donkey anti-mouse from Jackson ImmunoResearch Laboratories, Inc; and, Alexa Fluor 488–conjugated donkey anti-guinea pig from Molecular Probes.

Yeo C.T., Kropp E.M., Hansen P.A., Pereckas M., Oleson B.J., Naatz A., Stancill J.S., Ross K.A., Gundry R.L, & Corbett J.A. (2023). β-cell–selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake. The Journal of Biological Chemistry, 299(3), 102994.

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Immunoblotting and Immunofluorescence Antibody Protocols

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The antibodies listed below were used for immunoblotting (IB) or immunofluorescence (IF). Primary antibodies: mouse anti-CIP2A (clone 2G10-3B5; Santa Cruz sc80659, 1:500 IF, 1:1000 IB), rabbit anti-CIP2A (Cell Signalling Technologies #14805, 1:5000-12000 IB), rabbit anti-CIP2A (Novus Biologicals, NBP2-48710, 1:1000 IF), rabbit anti-phospho-Histone H2A.X (Ser139) (Cell Signalling Technologies #2577, 1:500 IF), mouse anti-phospho-Histone H2 A.X (Ser139) (clone JBW301; Millipore Sigma #05-636, 1:5000 IF), mouse anti-CHK1 (Santa Cruz sc8408, 1:500 IB), rabbit antiphospho-CHK1 (Ser345) (Cell Signalling Technologies #2348, 1:1000 IB), rabbit anti-KAP1 (Bethyl ON-TARGET Plus KIAA1524 (CIP2A) SMARTpool L-014135-01-0005, siGENOME MDC1 SMARTpool M-003506-04-0005, siGENOME TOPBP1 SMARTpool M-012358-01-0005).

Adam S., Rossi S.E., Moatti N., De Marco Zompit M., Xue Y., Ng T.F., Álvarez-Quilón A., Desjardins J., Bhaskaran V., Martino G., Setiaputra D., Noordermeer S.M., Ohsumi T.K., Hustedt N., Szilard R.K., Chaudhary N., Munro M., Veloso A., Melo H., Yin S.Y., Papp R., Young J.T.F., Zinda M., Stucki M, & Durocher D. (2021). The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer. Nature cancer, 2(12).

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