Ligase 4

Manufactured by Abcam

Sourced in United Kingdom

Ligase 4 is an enzyme that catalyzes the joining of DNA fragments through the formation of phosphodiester bonds. It plays a crucial role in the non-homologous end-joining (NHEJ) pathway, which is a major mechanism for the repair of DNA double-strand breaks.

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Lab products found in correlation

Dna pkcs, by Abcam (3 mentions) Tubulin, by Merck Group (2 mentions) Anti mouse hrp, by GE Healthcare (2 mentions) Rabbit flag, by Merck Group (2 mentions) Brdu a488 3d4, by BD (2 mentions) Ligase 3, by BD (2 mentions) Anti rabbit hrp, by GE Healthcare (2 mentions) G box imager, by Syngene (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Avantor (1 mentions) Xrcc4, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Selleck Chemicals (1 mentions) Artemis, by Abcam (1 mentions) Novex 3 8 tris acetate 15 well mini gels, by Thermo Fisher Scientific (1 mentions) Phospho dna pkcs s2056, by Abcam (1 mentions) Immobilon p membrane, by Merck Group (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions) Dna pkcs, by Santa Cruz Biotechnology (1 mentions)

4 protocols using ligase 4

Immunoblotting Protocol for Protein Analysis

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Immunoblotting was performed as described 7 (link) and developed using a Syngene G-Box imager. Antibodies: TRF2 (Karlseder lab), Rabbit FLAG (Sigma-Aldrich - F7425), FLAG (M2, Sigma-Aldrich - F1804), Tubulin (Sigma-Aldrich – T6557), BrdU A488 (3D4, BD Biosciences - 555627), Ku70 (V540, Cell Signalling - 4104), Ku70 (Abcam – ab3114), Ku86 (Cell Signalling - 2753), ATM (Epitomics - 1549-1), Ligase 4 (EPR16531, Abcam - ab193353), DNA-PKcs (Abcam - ab70250), Ligase 3 (BD Biosciences - 611876), Anti-Rabbit HRP (GE Healthcare - NXA931), Anti-Mouse HRP (GE Healthcare - NXA934V).

Arnoult N., Correia A., Ma J., Merlo A., Garcia-Gomez S., Maric M., Tognetti M., Benner C.W., Boulton S.J., Saghatelian A, & Karlseder J. (2017). Regulation of DNA Repair pathway choice in S/G2 by the NHEJ inhibitor CYREN. Nature, 549(7673), 548-552.

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Immunoblotting Protocol for Protein Analysis

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Western Blot Analysis of DNA Damage Repair Proteins

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After being washed with ice-cold PBS, cells were lysed with RIPA Lysis Buffer (VWR Life Science, Philadelphia, PA, USA), supplemented with Phosphatase Inhibitor Cocktail 2 (Sigma, St. Louis, MO, USA) and Protease Inhibitor Cocktail (Bimake, Houston, TX, USA). The Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was used to determine protein concentration. Equal protein lysates were run on Novex 3–8% Tris-acetate 15 Well Mini Gels (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon-P membranes (Millipore, Burlington, MA, USA). Membranes were then probed with the following primary antibodies: GAPDH (Santa Cruz Biotechnologies, Dallas, TX, USA), DNA-PKcs (abcam, Cambridge, UK), phospho DNA-PKcs S2056 (abcam), Artemis (abcam), phospho Artemis S516 (abcam), XRCC4 (Santa Cruz Biotechnologies), Ligase IV (abcam), CD4 (abcam), and DYKDDDDK (FLAG) Tag (Invitrogen). After exposure to the matching HRP-conjugated secondary antibody, cells were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).

Hu C., Bugbee T., Gamez M, & Wallace N.A. (2020). Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity. Cancers, 12(9), 2356.

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Quantitative Western Blot Analysis

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Western blotting was assessed as previously described [46 (link)]. Briefly, 40µg of total protein from each samples was loaded and resolved by electrophoresis in 3-8% SDS-PAGE gradient gels (Biorad), and transferred to nitrocellulose membrane (Hybond C Membrane (GE Healthcare). Blots were then incubated using appropriate antibodies: DNA-PK_cs, [1:500, at 4°C, overnight (ON) (SantaCruz Biotechnology)]; Ku70 [1:800, at 4°C, ON (abcam, UK)]; Ku80 [1:800, at 4°C, ON (abcam, UK)]; XRCC4 [1:1000, at 4°C, ON (AbDSerotec, UK)]; Ligase IV [1:800, at 4°C, ON (Abcam, UK)]; GAPDH [1:3000, at room temperature (RT), for 1 hr (Santa Cruz)]. Followed by HRP-conjugated, Goat anti-rabbit or Goat anti-mouse IgG-HRP secondary antibody [1:1000 at RT, for 1 hr, (Dako, Cambridge, UK)]. Image capture and analysis was carried out using the Fuji LAS-300 Image Analyser System (FujiFilm).

McCormick A., Donoghue P., Dixon M., O’Sullivan R., O’Donnell R.L., Murray J., Kaufmann A., Curtin N.J, & Edmondson R.J. (2016). Ovarian Cancers Harbour Defects in Non-Homologous End Joining Resulting in Resistance to Rucaparib. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(8), 2050-2060.

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