Ab15083

Manufactured by Abcam

Sourced in United Kingdom

Ab15083 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory techniques, including immunoassays and immunohistochemistry. The product details and technical specifications are available on the Abcam website.

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Lab products found in correlation

26 protocols using ab15083

Antibodies for Chromatin Remodeling

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Rabbit polyclonal antibodies Arp5 (ab12099), Yaf9 (ab4468), Eaf3 (ab4467), and H2A-S129phos (γH2AX; ab15083) are commercially available from Abcam, as is the AID antibody from BioRois (APC004Am). Anti-Snf6 and anti-Swi2, and Anti-Sth1 antibodies were kind gifts from J. Reese (Pennsylvania State University) and B. Cairns (University of Utah), respectively.

Bennett G, & Peterson C.L. (2015). SWI/SNF recruitment to a DNA double-strand break by the NuA4 and Gcn5 histone acetyltransferases. DNA repair, 30, 38-45.

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Chromatin Immunoprecipitation (ChIP-seq) Protocol

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ChIP-seq was performed as previously described (Can et al. 2019 (link)). Antibodies for IP were anti-H2A (39945, Actif motif), IgG (AB27478, Abcam), or anti- γH2A (AB15083, Abcam) or anti-GFP (3h9, Chromotek).

Morafraile E.C., Hänni C., Allen G., Zeisner T., Clarke C., Johnson M.C., Santos M.M., Carroll L., Minchell N.E., Baxter J., Banks P., Lydall D, & Zegerman P. (2019). Checkpoint inhibition of origin firing prevents DNA topological stress. Genes & Development, 33(21-22), 1539-1554.

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ChIP Assay for Histone Modifications

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ChIP was performed as described previously [12 (link)]. Samples were immunoprecipitated with 2 μl anti-Red1 serum (Lot#16440, kind gift of N. Hollingsworth) or 2 μl anti-phospho-H2A-S129 antibody (Abcam #ab15083) per sample. Library preparation was performed as described [15 ]. Library quality was confirmed by Qubit HS assay and 2200 Tape Station. 100-bp single-end sequencing was performed on an Illumina NextSeq 500 instrument.

Vale-Silva L.A., Markowitz T.E, & Hochwagen A. (2019). SNP-ChIP: a versatile and tag-free method to quantify changes in protein binding across the genome. BMC Genomics, 20, 54.

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Quantifying γ-H2AX Levels at DNA Double-Strand Breaks

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ChIP was carried out according to the protocol of Shroff et al. (9 (link)) using γ-H2AX antibody (abcam ab15083). γ-H2AX levels around the DSB were assessed by qPCR using primer sequences listed in SI Appendix, Table S5. The qPCR of immunoprecipitated samples was carried out using a Rotor-Gene SYBR Green PCR Kit (Qiagen 204076) in a Qiagen Rotor-Gene Q real-time PCR machine. γ-H2AX levels at all distances around the break were normalized to input as measured at the PHO5 locus with primers MT101 and MT102.

Li K., Bronk G., Kondev J, & Haber J.E. (2020). Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break. Proceedings of the National Academy of Sciences of the United States of America, 117(35), 21354-21363.

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Quantifying DNA Damage at CAG Repeats

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Strain YJK146 containing (CAG)₁₄₀ and the no repeat control YJK154 was analyzed by chromatin immunoprecipitation (ChIP) using antibodies recognizing phosphorylated H2A Serine129 (ab15083, Abcam). This modification has been referred to as γ-H2AX in yeast⁶⁵. Yeast were grown to an O.D. of ~0.8 in YPGal, arrested in G1 using alpha factor, then washed twice with water and released into YPGal. 50 mL cultures of 0, 20, 40, 60, and 90 minute time points were cross-linked with 1% formaldehyde for 15 minutes at room temperature, and quenched with glycine. Cells were lysed mechanically with 0.5 mm glass beads. The chromatin-containing cell suspension was sonicated to yield sheared DNA in the range of approximately 100–1500 bp. Antibody bound DNA was immunoprecipitated (IP) using Protein G Dynabeads (Invitrogen). Samples were analyzed by quantitative PCR using QuantStudio 6 (Applied Biosystems). Relative quantities of IP and input DNA were determined using a standard curve for primers at the CAG locus as well as non-enriched control, ACT1. IP/input values for the CAG locus is normalized to ACT1. The graph shows the fold-enrichment of the CAG strain compared to the no repeat control strain.

Kim J.C., Harris S.T., Dinter T., Shah K.A, & Mirkin S.M. (2016). The role of break-induced replication in large-scale expansions of (CAG)n•(CTG)n repeats. Nature structural & molecular biology, 24(1), 55-60.

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Chromatin Immunoprecipitation and DNA Damage Sensitivity Assay

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Chromatin immunoprecipitation assay was carried out as described [11] (link), [61] (link). The DNA damage sensitivity assay was determined as described [61] (link). Immunoblotting was described previously [61] (link). UV and MMS synchrony experiments were carried out as described previously [7] (link), [27] (link). Plasmid shuffle with 5-fluoroorotic acid (5-FOA) was performed as described previously [62] (link), [63] (link). Rabbit anti-GFP antibody (ab290) and anti-phospho histone H2A antibody (ab15083) were purchased from Abcam (Cambridge, MA). Rabbit anti-H2A antibody (39235) was obtained from Active Motif (Carlsbad, CA).

Bandhu A., Kang J., Fukunaga K., Goto G, & Sugimoto K. (2014). Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism. PLoS Genetics, 10(2), e1004136.

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Chromatin Immunoprecipitation Assay

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Anti-γH2A (phospho-S129) and anti-H3K9me3 antibodies (ab15083 and ab8898, Abcam) were used to perform immunoprecipitation assays. The wild-type extract was used as control. qPCR was carried out to quantify the levels of DNA in the precipitates. The relative DNA-binding levels of ChIP assays were determined by comparing the relative enrichment of the chromatin-immunoprecipitated DNA between the quelled strain and a wild-type strain. Relative enrichment of the chromatin-immunoprecipitated DNA was normalized to the actual al-1 copy numbers in each quelled strain or transformant. actin levels in the chromatin-immunoprecipitated DNA were used to normalize the loading in different samples.

Yang Q., Ye Q.A, & Liu Y. (2015). Mechanism of siRNA production from repetitive DNA. Genes & Development, 29(5), 526-537.

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Protein Detection by Western Blot

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For protein detection by western blot, 2 × 10⁷ cells were harvested at the indicated time points and snap-frozen. Protein lysates were prepared by alkaline lysis and subsequent trichloroacetic acid precipitation. For analysis of γH2A, samples were run on pre-cast NuPage gels (4–12% Bis-Tris, Invitrogen) using MES buffer for 35 min at 200 V. To detect checkpoint activation by analysis of the Rad53 phosphorylation shift, samples were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for 180 min at 160 V. Western blotting was performed with standard methods. The γH2A phosphorylation was detected using anti-γH2A (ab15083, Abcam; 1:3000) antibody, for Rad53 shift detection anti-Rad53 (ab104232, Abcam; 1:4000) was used. As loading control, the membranes were washed and re-incubated with anti-Pgk1 antibody (22D5C8, Invitrogen; 1:7000) or anti-Cdc48 antibody (lab of Stefan Jentsch; 1:10,000). Unprocessed scans of all main figures and western blots can be found in the Source data file.

Bantele S.C., Lisby M, & Pfander B. (2019). Quantitative sensing and signalling of single-stranded DNA during the DNA damage response. Nature Communications, 10, 944.

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Rad53, H2A, and γH2A Detection

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Detection of Rad53 was performed using ab104232 (Abcam, 1:5000), H2A with ab13923 (Abcam, 1:1000), and γH2A (phospho S129) with ab15083 (Abcam, 1:1000).

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TCA Protein Extraction and Western Blot

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Total cell lysates were prepared by TCA protein extraction method. Cells were resuspended in TCA buffer (1.85 M NaOH and 7.4% β-mercaptoethanol) for 10 min and then mixed with equal volume of 20% trichloroacetic acid to precipitate lysates. Cell lysates were dissolved in 0.1% NaOH and resolved using SDS-PAGE, then transferred onto PVDF membranes (Immobilon^®-P, Millipore); membranes were then incubated with the indicated antibodies. The following antibodies were used in this study (dilutions used, source and catalog numbers): α-H2A S129-P (1:2000, Abcam, ab15083), α-H3K4me3 (1:10,000, Abcam, ab8580), α-H3K4me2 (1:5000 Abcam, ab7766), α-H3K4me1 (1:2000, Abcam, ab8895), α-HA (1:5000, Roche, 11867423001) and α-G6PDH (1:20,000, Sigma, A9521). Signals were detected using the ECL detection reagent (Millipore Immobilon™) with UVP BioSpectrum^® imaging system. Uncropped blots can be found in Source data.

Chong S.Y., Cutler S., Lin J.J., Tsai C.H., Tsai H.K., Biggins S., Tsukiyama T., Lo Y.C, & Kao C.F. (2020). H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress. Nature Communications, 11, 809.

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