Sc 22760

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-22760 is a lab equipment product offered by Santa Cruz Biotechnology. It is designed for use in scientific research applications.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

53BP1 (SC-22760, (5 citations) Anti-53BP1 (sc-22760; (2 citations) Anti-53BP1 (H-300) (sc-22760, (2 citations) Rabbit anti-53BP1(#sc-22760) (2 citations)

16 protocols using sc 22760

Quantifying DNA Damage Response via 53BP1 Kinetics

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

IR-induced 53BP1 kinetics were determined as previously outlined with modifications (19 (link)). Cells were grown on coverslips one day before the experiment and on the day of the experiment, cells were exposed to 1Gy of γ-rays. At different time points after IR (see figure), cells were washed twice with ice cold 1× PBS and fixed with 4% paraformaldehyde (in 1× PBS) for 20 min at RT, washed 5 times with 1× PBS, and incubated in 0.5% Triton X-100 on ice for 10 min. Cells were washed 5 times with 1× PBS and incubated in blocking solution (5% goat serum (Jackson Immuno Research) in 1× PBS) overnight. The blocking solution was then replaced with the 53BP1 (SC-22760, Santa Cruz) primary antibody diluted in 5% goat serum in 1x PBS and the cells were incubated for 2 h. Cells were then washed 5 times with wash buffer (1% BSA in 1× PBS). Cells were incubated with the Alexa Fluor 488 (1:1000) (Molecular Probes) secondary antibody in 1% BSA, 2.5% goat serum in 1× PBS for 1 h in the dark, followed by five washes. After the last wash, cells were mounted in VectaShield mounting medium containing DAPI. The images were acquired using a Zeiss Axio Imager fluorescence microscope utilizing a 63× oil objective. ≥100 cells were analyzed for each time point.

Saha J., Bae J., Wang S.Y., Lu H., Chappell L.J., Gopal P, & Davis A.J. (2021). Ablating putative Ku70 phosphorylation sites results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma. Nucleic Acids Research, 49(17), 9836-9850.

+ Open protocol

+ Expand

Western Blot and Immunoprecipitation Antibodies

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used for western blot analysis: anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab) [1 (link)], anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz, Dallas, TX, USA), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-Histone3 rabbit polyclonal antibody (ab1791, Abcam, Cambridge, UK), and anti-α-Tubulin mouse monoclonal antibody (sc-5286, Santa Cruz). For immunoprecipitation assay, anti-HA rabbit polyclonal antibody (F-7, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab), and anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz) were used. The following antibodies were used for immunofluorescence staining: anti-MDC1 rabbit polyclonal antibody (R2), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-RNF8 goat polyclonal antibody (ab15850, Abcam), anti-53BP1 rabbit polyclonal antibody (sc-22760, Santa Cruz), anti-BRCA1 mouse monoclonal antibody (sc-6954, Santa cruz), anti-γH2AX mouse monoclonal antibody (05-636-1, Millipore, Burlington, MA, USA), and anti-RNF168 rabbit polyclonal antibody (ABE367, Millipore).

Radhakrishnan K., Park S.J., Kim S.W., Hariharasudhan G., Jeong S.Y., Chang I.Y, & Lee J.H. (2020). Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1. International Journal of Molecular Sciences, 21(7), 2650.

+ Open protocol

+ Expand

Antibody Utilization for Immunoblot and Immunofluorescence

Check if the same lab product or an alternative is used in the 5 most similar protocols

All the antibodies are listed in Table 1. These antibodies, used in immunoblots and/or immunofluorescence assays, were diluted in TBS-0.1% Tween20 or PBS-1% BSA, respectively.Table 1

List of antibodies and conditions used in this work

Antibody		Dilution (WB/IF)	Clone and/or reference	Supplier
53BP1	Rabbit polyclonal	-; 1/200	H300, sc-22,760	Santa Cruz Biotechnology
53BP1	Rabbit polyclonal	-; 1/200	NB100–304	Novus Biologicals
Histone H4-K16 ac	Rabbit monoclonal	1/500; 1/400	ab109463	Abcam
Nbs1	Mouse monoclonal	-; 1/200	611,871	BD Biosciences
VRK1	Mouse monoclonal	1/1000; 1/200	1B5	Own production [74 (link)]
VRK1	Rabbit polyclonal	1/1000;-	VC	Own production [74 (link)]
VRK1 (N-term)	Rabbit polyclonal	-; 1/200	HPA000660	Sigma-Aldrich
β-actin	Mouse monoclonal	1/2000; −	AC15/A5441	Sigma-Aldrich
γH2AX	Mouse monoclonal	-; 1/200	Clone JBW301; 05–636	Millipore
Anti-mouse IgG (WB)	Goat Anti-Mouse IgG, DyLight 680 (red)	1/10000; −	35,518	Thermo Scientific
anti-rabbit IgG (WB)	Goat anti-rabbit IgG, DyLight 800 (green)	1/10000; −	35,571	Thermo Scientific
Goat anti-Mouse IgG (IF)	Goat anti-Mouse IgG linked to Cy3 (red)	-; 1/1000	115–165-146	Jackson ImmunoResearch; West Grove, PA, USA
Goat anti-rabbit IgG (IF)	Goat anti-rabbit IgG linked to Cy2 (green)	-; 1/1000	111–225-144	Jackson ImmunoResearch; West Grove, PA, USA

Campillo-Marcos I, & Lazo P.A. (2019). Olaparib and ionizing radiation trigger a cooperative DNA-damage repair response that is impaired by depletion of the VRK1 chromatin kinase. Journal of Experimental & Clinical Cancer Research : CR, 38, 203.

+ Open protocol

+ Expand

Quantifying DNA Damage Response in Ras-Overexpressing Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For deoxynucleosides experiments, overexpression of Ras was induced by incubation with Dox for 8 days. Where indicated, cells were treated with the mix of deoxynucleosides (50 nM of dA, dU, dC and dG) for the whole course of the experiment (Bester et al., 2011) or with 50 μM of cordycepin for the last 2 h of the experiment (Jones et al., 2013). Cells were then fixed with 4% formaldehyde (15 min RT) and permeabilized with 0.1% Triton‐X‐100 (15 min RT). Samples were co‐immunostained with antibodies against 53BP1 (rabbit, sc‐22760, Santa Cruz, 1:1000) and cyclin A (mouse, NCL‐CYCLIN A, Leica‐Novocastra, 1:50), followed by secondary antibodies AlexaFluor568 goat anti‐rabbit and AlexaFluor488 goat anti‐mouse, respectively. To detect γH2AX we used the mouse anti‐γH2AX antibody (613402, Biolegend, 1:500). 250 non‐overlapping images were acquired for each cells‐containing coverslip using the Olympus Scan‐R microscope. At least 4000 cells were scored and images were processed using Scan‐R Analysis software. We scored the total intensity of the pan‐nuclear γH2AX signal in individual nuclei to avoid the variation within an asynchronous cell population (Mistrik et al., 2009; Toledo et al., 2013). For the 53BP1 foci analysis, only cyclin A negative cells were scored. Differences were analyzed using the Wilcoxon signed‐rank test.

Maya-Mendoza A., Ostrakova J., Kosar M., Hall A., Duskova P., Mistrik M., Merchut-Maya J.M., Hodny Z., Bartkova J., Christensen C, & Bartek J. (2014). Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress. Molecular Oncology, 9(3), 601-616.

+ Open protocol

+ Expand

DDR Signaling Assay in U-2 OS Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

U-2 OS cells were grown in 96-well flat bottom tissue culture treated microplates (Falcon®, 353219), treated with compounds at the indicated concentrations for 3 hours before irradiation with 10Gy (X-RAD 320) according to manufacturer’s instructions. Cells were then incubated for 3 hours before fixed in 3.7% formaldehyde for 20 minutes, incubated for 5 minutes with cell permeabilisation buffer (0.25% Triton-X (v/v) in PBS) at room temperature. For fluorescence staining, cells were next blocked in 10% FBS for 40 minutes prior to incubation with either anti-Mdc1 (Abcam, ab11171) or anti-53bp1 (Santa Cruz, sc-22760) primary antibodies (in 1% FBS, 0.05% Triton-X (v/v)) for 1 hour at room temperature. Alexa Fluor® 488 conjugate secondary antibody (Life Technologies, A-11008) was then added for an additional hour at room temperature in the dark before staining Hoechst 33342 (Life Technologies, R37605). Images were acquired and captured using the LSM-710 fluorescent microscope.

Maculins T., Carter N., Dorval T., Hudson K., Nissink J.W., Hay R.T, & Alwan H. (2016). A Generic Platform for Cellular Screening Against Ubiquitin Ligases. Scientific Reports, 6, 18940.

+ Open protocol

+ Expand

Immunofluorescence Staining of DNA Damage Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were grown on glass coverslips, washed with PBS and fixed with 4% formaldehyde for 10 min. After washing with PBS, cells were incubated with methanol at −20 °C for 10 min. Cells were then washed twice with PBS and incubated with primary antibodies for 1 h at RT. Following the washing step, coverslips were incubated with anti-rabbit or anti-mouse AlexaFluor-488 or -568 secondary antibodies (Invitrogen) with DAPI (Invitrogen) for 1 h at RT, washed again with PBS and mounted using Prolong Gold Antifade (Thermo Fisher Scientific). The primary antibodies used: LC3B (D11) XP™ (1:200, 3868, Cell Signalling), gamma H2A.X (phospho-S140) (1:1000, ab22551, Abcam), 53BP1 (H300) (1:500, sc-22760, Santa Cruz), TOMM20 (1:1000, ab56783, Abcam).

Vanzo R., Bartkova J., Merchut-Maya J.M., Hall A., Bouchal J., Dyrskjøt L., Frankel L.B., Gorgoulis V., Maya-Mendoza A., Jäättelä M, & Bartek J. (2019). Autophagy role(s) in response to oncogenes and DNA replication stress. Cell Death and Differentiation, 27(3), 1134-1153.

+ Open protocol

+ Expand

Western Blotting and Immunofluorescence Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For Western blotting shown in Figure 1C and D, Figure 1—figure supplement 1 and Figure 3—figure supplement 1C we used the following primary antibodies: rabbit anti-H3 (Ab1791, Abcam – RRID:AB_302613), mouse anti-MBP (E8032, NEB – RRID:AB_1559732), rabbit anti-H2A (raised against amino acid residues 100–130)(Fradet-Turcotte et al., 2013 (link)), and rabbit anti-H2A (targeting 719 the acidic patch, 07–146, Millipore – RIDD:AB_310394). Peroxidase-affiniPure goat anti-rabbit IgG (111 035 144, Jackson Immuno Research – RRID:AB_2307391) and HRP-linked sheep anti-mouse IgG (NA931, GE Healthcare – RIDD:AB_772210) were used as secondary antibodies. For immunofluorescence and FACS analyses, as shown in Figure 5—figure supplement 2, cells were stained for mouse anti-γ-H2AX (clone JBW301, Millipore– RIDD:AB_309864) and rabbit anti-53-BP1 (sc-22760, Santa Cruz – RIDD:AB_2256326). The following antibodies were used as secondary antibodies in immunofluorescence microscopy: Alexa Fluor 555 anti-rabbit and AlexaFluor 647 goat anti-mouse (Molecular Probes – RIDD:AB_141784 and RIDD:AB_141725, respectively). DNA was counterstained with DAPI to trace the outline of nuclei.

Kitevski-LeBlanc J., Fradet-Turcotte A., Kukic P., Wilson M.D., Portella G., Yuwen T., Panier S., Duan S., Canny M.D., van Ingen H., Arrowsmith C.H., Rubinstein J.L., Vendruscolo M., Durocher D, & Kay L.E. (2017). The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage. eLife, 6, e23872.

+ Open protocol

+ Expand

Antibody and Reagent Usage for DNA Damage Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used in our studies: mouse monoclonal anti-Flag (1:10000, F3165, Sigma-Aldrich), mouse monoclonal anti-HA (1:10 000, H9658, Sigma-Aldrich), rabbit polyclonal anti-53BP1 (IF 1:200, sc-22760, Santa Cruz Biotechnology), rabbit polyclonal anti-MDC1 (1:1000, ab11171, Abcam), rabbit polyclonal anti-BRCA1 (IF 1:100, BS6423, Bioworld), polyclonal anti-γH2AX (IF 1:200, BS4760, Bioworld), rabbit polyclonal anti-Histone H3 (1:10 000, BE3015, EASYBIO), anti-β-Tubulin (1:10 000, BE3212-10, EASYBIO), rabbit polyclonal anti-ESCO2 (1:1000, A301-689A, Bethyl), and mouse monoclonal anti-acetyl-SMC3 K105/106, Clone 21A7 (ChIP 2μg, MABE1073, Millipore). The following reagents were used in our studies: KU55933 (ATM kinase inhibitor, S1092, Selleck Chemicals), NU7441 (DNA-PK inhibitor, S2638, Selleck Chemicals), VE821 (ATR inhibitor, S8007, Selleck Chemicals), DNase I (D5025, Sigma) and bleomycin sulfate (HY-17565, MedChemExpress).

Fu J., Zhou S., Xu H., Liao L., Shen H., Du P, & Zheng X. (2023). ATM–ESCO2–SMC3 axis promotes 53BP1 recruitment in response to DNA damage and safeguards genome integrity by stabilizing cohesin complex. Nucleic Acids Research, 51(14), 7376-7391.

+ Open protocol

+ Expand

High-Throughput Screening of DNA Damage Response

Check if the same lab product or an alternative is used in the 5 most similar protocols

The compound library was obtained from the National Cancer Institute and was composed of 933 compounds including natural product compounds and bioactive compounds (Natural Compounds Set III and 1 mM Mechanistic Diversity Set II). Monoclonal antibodies for western blotting against phosphorylated RPA (p-RPA),RPA,and CTIP were purchased from Bethyl (Montgomery,TX); anti-p-CHK1 and anti-CHK1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA); anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas,TX);and anti-tubulin antibody was purchased from Sigma (St. Louis, MO). Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology, Inc. Primary antibody for immunofluorescent staining against Rad51 (SC-8349) and 53BP1 (SC-22760) were purchased from Santa Cruz Biotechnology, Inc., anti-p-RPA32 (A300-245A)was purchased from Bethyl, anti-γ-H2AX (16-202A) was purchased from EMD Millipore. Secondary antibody Alex Fluor-conjugated goat anti-rabbit IgG (A21206) and secondary antibody Alex Fluor-conjugated goat anti-mouse IgG (A-11001) were purchased from Life Technology (Waltham, MA).

Zhang L., Peng Y., Uray I.P., Shen J., Wang L., Peng X., Brown P.H., Tu W, & Peng G. (2017). Natural product β-thujaplicin inhibits homologous recombination repair and sensitizes cancer cells to radiation therapy. DNA repair, 60, 89-101.

+ Open protocol

+ Expand

Immunohistochemistry and Immunofluorescence Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following primary antibodies were adopted for IHC and IF on mouse and human samples: rabbit polyclonal ACE2 (1:500 pH8, ab15348, Abcam), rabbit polyclonal Prosurfactant Protein C (1:200 pH9, AB3786, Merck Millipore), mouse monoclonal TTF‐1 (clone SPT24, 1:100 pH6, NCL‐L‐TTF‐1, Leica Novocastra), rabbit polyclonal anti CD31 (1:50 pH9, ab28364, Abcam), rabbit polyclonal anti‐53BP1 (1:1,000, sc‐22760, Santa Cruz), and mouse monoclonal anti‐phospho‐Histone H2A.X (1:500, 05‐636, Sigma‐Aldrich).

Sepe S., Rossiello F., Cancila V., Iannelli F., Matti V., Cicio G., Cabrini M., Marinelli E., Alabi B.R., di Lillo A., Di Napoli A., Shay J.W., Tripodo C, & d’Adda di Fagagna F. (2021). DNA damage response at telomeres boosts the transcription of SARS‐CoV‐2 receptor ACE2 during aging. EMBO Reports, 23(2), e53658.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!