Cells grown on glass cover slips were fixed in 4% formaldehyde supplemented with 0.1% Triton X100 for 10 min at room temperature before permeabilization in 0.5% Triton X100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies against FANCD2 (ab2187, Abcam), PhosphoDNA-PKcs (Ser2056 ab18192, Abcam), MDC1 (ab11169, Abcam), γH2AX (JBW301, Upstate), 53BP1 (MAB3802, Millipore), Mre11 (GTX70212, GeneTex), Rad51 (PC130, Calbiochem), RIF1 (A300–569A, Bethyl), RAP80 (NBP1–87156, Novus) and acetylated lysine (MA1–2021, Thermo). Primary antibody detection was achieved by incubation with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 secondary antibodies (Invitrogen) for 30 min at room temperature. Slides were mounted in DAKO mounting medium supplemented with DAPI (Sigma) and examined at a magnification of 63x using a fluorescence microscope (Zeiss Axio Observer Z1) equipped with an ORCA-ER camera (Hamamatsu). The microscope and camera parameters were set for each series of experiments to avoid signal saturation. Image processing and analysis were performed using ImageJ software (http://rsb.info.nih.gov/ij/ ).
Nbp1 87156
Manufactured by Novus Biologicals
NBP1–87156 is a laboratory reagent product. It serves as a detection antibody for use in immunodetection applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab2187, by Abcam (1 mentions)
Alexa fluor 488 or 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Hamamatsu Photonics (1 mentions)
Ma1 2021, by Thermo Fisher Scientific (1 mentions)
Ab11169, by Abcam (1 mentions)
Mab3802, by Merck Group (1 mentions)
Gtx70212, by GeneTex (1 mentions)
Dako mounting medium, by Merck Group (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab108411, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Jbw301, by Merck Group (1 mentions)
Sc 751, by Santa Cruz Biotechnology (1 mentions)
A8592, by Merck Group (1 mentions)
G9545, by Merck Group (1 mentions)
Mms 101r, by Fortrea (1 mentions)
3 protocols using nbp1 87156
1
Immunofluorescence Staining of DNA Damage Markers
2
Western Blotting and Immunofluorescence Protocol
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The following antibodies were used for western blotting for human or mouse cells: Rabbit anti-human/mouse RAP80 (NBP1-87156; Novus Biologicals) at 1:1,000 for human cells, 1:100 for mouse cells; Rabbit anti-human RAP80 (A300-763A; Bethyl Laboratories) at 1:1,000. Rabbit anti-human MERIT40 (A302-515A; Bethyl Laboratories) at 1:1,000. Rabbit anti-human/mouse MERIT40 (D7Y5K; 12711S; Cell Signaling) at 1:1,000. Rabbit anti-human/mouse Abraxas1 (NBP1-22977; Novus Biologicals) at 1:1,000. Rabbit anti-mouse BRCC45 (EPR11858; ab177960; Abcam) at 1:1,000. Rabbit anti-human/mouse BRCC36 (ab108411; Abcam) at 1:50,000. Mouse anti-human P4D1 (sc-8017; Santa Cruz) at 1:1,000. Mouse anti-human GFP (sc-9996; Santa Cruz) at 1:500. Mouse anti-human/mouse Tubulin (3873S; Cell Signaling) at 1:10,000; Mouse anti-human Tubulin (12G10; DSHB) at 1:1,000. Mouse anti-human HA.11 (901502; BioLegend) at 1:1,000. The following antibodies were used for immunofluorescence experiment: Rabbit anti-mouse BRCA1 (homemade rabbit polyclonal antibody raised against the exon 11 region of mouse BRCA1) at 1:100; mouse anti-human BRCA1 (sc-6954; Santa Cruz) at 1:500; rabbit anti-mouse MERIT40 (polyclonal antibody raised against GST-MERIT40 as previously described [Shao et al., 2009 (link)]) at 1:200; mouse anti-mouse γH2AX (JBW301; Millipore) at 1:2,000; rabbit anti-human γH2AX (39117; Active Motif) at 1:500.
3
Immunofluorescence and Immunoblotting Antibodies
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We employed the following antibodies for immunofluorescence: mouse anti‐γH2AX (clone JBW301, Millipore, 1:1,000), mouse anti‐BRCA1 (clones MS110 and MS13 Calbiochem, 1:100), rabbit anti‐BRCA1 (#07‐434, Millipore 1:1,000), rabbit anti‐RAD51 serum (70‐001; lot 1; BioAcademia, 1:150,000), rabbit anti‐RAP80 rabbit (A300‐763A, Bethyl Laboratories Inc., 1:200), anti‐RAP80 rabbit (NBP1‐87156, Novus Biologicals, 1:500), rabbit anti‐ABRAXAS (A302‐180A, Bethyl Laboratories Inc., 1:500), rabbit anti‐cyclin A (sc‐751, Santa Cruz; 1:200), mouse anti‐HA.11 (MMS‐101R, Covance; 1:1,000), and rabbit anti‐pS4/S8 RPA32 (A300‐245A, Bethyl Laboratories Inc., 1:1,000) We employed the following antibodies for immunoblotting: rabbit anti‐BRCA1 (homemade, 1:1,000) (Noordermeer et al, 2018 (link)), rabbit anti‐RNF8 (kind gift from J. Chen, 1:2,500), rabbit anti‐RNF168 (homemade, 1:2,500) (Stewart et al, 2009 (link)), rabbit anti‐RAP80 (A300‐763A, Bethyl Laboratories Inc., 1:5,000), mouse anti‐tubulin (clone DM1A, Calbiochem, 1:1,000), rabbit anti‐KAP1 (Bethyl, 1:10,000), rabbit anti‐GAPDH (G9545, Sigma Aldrich, 1:5,000), mouse anti‐FLAG M2‐Peroxidase (HRP) (A8592, Sigma Aldrich, 1:1,000), and rabbit anti‐GFP (ab290, Abcam; 1:10,000).
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