Na19l

Manufactured by Merck Group

The NA19L is a laboratory equipment product from Merck Group. It is designed to perform specific functions within a laboratory setting. The core function of the NA19L is to facilitate laboratory processes, however, a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

4 protocols using na19l

Immunofluorescence Microscopy of RAD51 and RPA2

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Immunofluorescence was performed as previously described (5 (link)). Antibodies used were as follows: RAD51 (398587, 1:800, Santa Cruz Biotechnology) and RPA2 (NA19L, 1:500, Millipore, RRID: AB_565123). Cells were imaged using a Zeiss LSM510 confocal microscope or Keyence Fluorescence Microscope BZ-X710.

Nichols B.A., Oswald N.W., McMillan E.A., McGlynn K., Yan J., Kim M.S., Saha J., Mallipeddi P.L., LaDuke S.A., Villalobos P.A., Rodriguez-Canales J., Wistuba I.I., Posner B.A., Davis A.J., Minna J.D., MacMillan J.B, & Whitehurst A.W. (2018). HORMAD1 Is a Negative Prognostic Indicator in Lung Adenocarcinoma and Specifies Resistance to Oxidative and Genotoxic Stress. Cancer research, 78(21), 6196-6208.

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Antibody Characterization for Western Blotting and Immunofluorescence

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The following antibodies were used for Western blotting (WB) and/or immunofluorescence (IF). Anti-53BP1 (sc-22760, Santa Cruz, WB 1:500, IF 1:300), anti-γ-H2AX (JBW301, Millipore, WB 1:5000, IF 1:300), anti-γ-H2AX (ab11174, Abcam, IF 1:300), anti-Flag (clone M2, Sigma, WB 1:5000, IF 1:500), anti-β-tubulin (CST2146S, Cell Signaling, WB 1:5000), anti-GAPDH (sc-32233, Santa Cruz, WB 1:1000), anti-GFP (sc-9996, Santa Cruz, WB 1:1000, IF 1:100), anti-HA (sc-805, Santa Cruz, WB 1:1000), anti-Myc (CST2272, Cell Signaling, WB 1:1000, IF 1:100), anti-USP21 (HPA028397, Sigma, WB 1:500–1:1000), anti-BRCA2 (OP95, Calbiochem, WB 1:1000), anti-BRCA1 (sc-SC-6954, Santa Cruz, IF 1:100), anti-RAD51 (sc-8349, Santa Cruz, WB 1:1000, IF 1:50), anti-CtIP (sc-271339, Santa Cruz, IF 1:100), anti-RPA (NA19L, Millipore, 1:200), FK2 (BML-PW8810-0100, Enzo, IF 1:1000), anti-PALB2 (gift from B. Xia, WB 1:1000), anti-LaminA/C (sc-6215, Santa Cruz, WB 1:1000). The following secondary antibodies were used for WB: goat anti-rabbit HRP IgG (sc-2030, Santa Cruz, 1:5000), goat anti-mouse HRP IgG (sc-2031, Santa Cruz, 1:5000), donkey anti-goat HRP IgG (sc-2056, Santa Cruz, 1:1000). Secondary antibodies for IF were used at 1:250 dilution: Alex Fluor 488 anti-mouse IgG (H + L), Alexa Fluor 488 anti-Rabbit IgG (H + L), Alex Fluor 568 anti-mouse IgG (H + L), Alex Fluor 568 anti-Rabbit IgG (H + L).

Liu J., Kruswick A., Dang H., Tran A.D., Kwon S.M., Wang X.W, & Oberdoerffer P. (2017). Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth. Nature Communications, 8, 137.

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Western Blot Analysis of DNA Damage Markers

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Tissues and cells were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate). Total protein levels were measured using the BCA method (Thermo), and 40–80 μg of protein was loaded into 10–12% acrylamide gels. Proteins were transferred to PVDF membranes, blocked with 5% skim milk, and blotted with antibodies against AIMP3 (human atlas, 1:1000), γH2AX (cell signalling, 1:1000), phospho RPA(S4/S8) (Bethyl A300-245A, 1:1000), RPA (Millipore NA19L, 1:500), Chk1 (sc-8408, 1:500), phospho-Chk1 (cell signalling 2341, 1:1000), β-actin (Sigma AC-74, 1:10000), tubulin-α (sc-12462R, 1:1000), and hsp90 (Santa Cruz sc-7947, 1:1000) in 1% milk or 3% BSA in PBS. To detect multiple proteins, same samples were processed in parallel in different gels, or single transferred membranes were cut into pieces according to protein size for immunoblotting. Membranes were washed in PBST (0.03% Triton X) and blotted with HRP conjugated-antibodies against mouse or rabbit IgG (Thermo) in 5% skim milk. Membranes were developed using ECL reagent (Santa Cruz).

Kim D., Kim S., Oh Y., Park S., Jeon Y., Kim H., Lee H, & Kim S. (2018). AIMP3 Deletion Induces Acute Radiation Syndrome-like Phenotype in Mice. Scientific Reports, 8, 15025.

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Quantifying RPA Chromatin Recruitment

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The recruitment of RPA to chromatin was performed essentially as described^{47 (link)} with minor modifications. This protocol allows to quantify only the fraction of RPA that is associated to chromatin and to measure in parallel the cell cycle distribution using propidium iodide (PI) staining. Briefly, untreated or HU-treated cells were permeabilized with PBS/Triton-X 0.2% for 5 min on ice (non-chromatin-bound protein extraction step), washed with BSA 5%/PBS and then fixed with paraformaldehyde 4% for 10 min at room temperature. Cells were then resuspended in BSA 5%/PBS for 1 h, incubated with primary RPA32 antibody (1:100, NA19L, Millipore) for 2 h at room temperature and then with Alexa Fluor 488-conjugated secondary goat anti-mouse antibody (Thermo Fisher Scientific) for 1 h in the dark. Finally, DNA was stained with PI (Sigma) for 20 min at room temperature in the dark. More than 25,000 cells for every sample were acquired using CellQuest software on FACSCalibur instrument and analyzed using FlowJo.

Menolfi D., Jiang W., Lee B.J., Moiseeva T., Shao Z., Estes V., Frattini M.G., Bakkenist C.J, & Zha S. (2018). Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA. Nature Communications, 9, 5351.

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