Anti phospho h3ser10

Manufactured by Merck Group

Anti-phospho-H3Ser10 is a laboratory reagent used to detect and quantify the phosphorylation of histone H3 at serine 10. This modification is an important epigenetic marker associated with various cellular processes, including chromosome condensation during mitosis and meiosis. The product can be used in techniques such as Western blotting, immunocytochemistry, and flow cytometry to analyze the levels of this modification in cellular samples.

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4 protocols using anti phospho h3ser10

Plk1 Regulation of Pentose Phosphate Pathway

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Antibodies and reagents used in the study are listed as following: anti-Plk1 (Sigma-Aldrich, P5998, 1:10,000), anti-Flag (Sigma, F3165, 1:5000), anti-HA (Sigma, H9658, 1:5000), anti-G6PD (Protein-tech, 25413-1-AP, 1:1000), anti-G6PD (Abcam, ab993, 1:1000), anti-glutathione S-transferase (anti-GST; Protein-tech, 10000-0-AP, 1:5000), anti-phospho-H3Ser10 (Millipore, 06-570, 1:1000), anti-cyclin B1 (Protein-tech, 55004-1-AP, 1:1000), anti-phospho-threonine (Millipore, AB1607, 1:1000), anti-PGLS (Santa Cruz, SC-398833, 1:1000), anti-6PGD (Protein-tech, 14718-1-AP, 1:1000), anti-CDK2 (Protein-tech, 10122-1-AP, 1:1000), rabbit IgG (Protein-tech), mouse IgG (Santa Cruz), and anti-Actin (Protein-tech, 60008-1-Ig, 1:5000). NADP⁺, NADPH, GSH, G6P, 6PG, NAC, Dox, tetracycline, HU, propidium iodide, nocodazole, 4,6-diamidino-2-phenylindole (DAPI), casein, D₂O, ribonucleosides, and deoxyribonucleosides were purchased from Sigma-Aldrich; R5P, nucleotides (AXP, GXP, CXP, and UXP, X = M, D, and T) were purchased from Sangon Biotech. BI2536 was purchased from Selleck Corporation, PP2A was from Millipore, and human recombinant Plk1 was from Sino biological company. [2-¹³C]-glucose and [U-¹³C]-glucose were from Cambridge Isotope Laboratories. γ-³²P ATP was from China Isotope & Radiation Corporation.

Ma X., Wang L., Huang D., Li Y., Yang D., Li T., Li F., Sun L., Wei H., He K., Yu F., Zhao D., Hu L., Xing S., Liu Z., Li K., Guo J., Yang Z., Pan X., Li A., Shi Y., Wang J., Gao P, & Zhang H. (2017). Polo-like kinase 1 coordinates biosynthesis during cell cycle progression by directly activating pentose phosphate pathway. Nature Communications, 8, 1506.

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Quantifying Cellular DNA Damage Response

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Cells were either irradiated with 10 Gy IR or left untreated. Thereafter, cells were fixed in 70% ethanol and stored at – 20° C. Cells were washed with PBS, incubated in PBS/0.25 % Triton X-100 on ice for 15 min followed by a subsequent incubation with anti-phospho-H3 (Ser 10) (06-570, Millipore, Billerica, MA) antibody and goat anti-rabbit IgG FITC conjugated antibody (111-096-144, Jackson ImmunoResearch Labs, West Grove, PA) in PBS + 1% BSA. Cells were washed and incubated with 10 μg/ml PI (propidium iodide) and 250 μg/ml RNAse A and analyzed by FACSCalibur (BD, Franklin Lakes, NJ). Data was assessed by the WinMDI 2.9 software.

Goldstein M, & Kastan M.B. (2015). Repair versus checkpoint functions of Brca1 are differentially regulated by site of chromatin binding. Cancer research, 75(13), 2699-2707.

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Quantitative Western Blot Analysis

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Egg extracts were collected by aspiration from microfluidics devices following the indicated experimental treatments. For each condition, 0.5 µl of extract was diluted in 10 µl of 3X Laemmli sample buffer (Bio-Rad) ^{77 (link)} and boiled at 95°C for 5 min. Equal volumes of samples were loaded into wells of a 4–15% Gradient SDS-PAGE gel (Bio-Rad) along with a protein standards (Bio-Rad) and separated by protein gel electrophoresis apparatus (BioRad). The proteins were then transferred from the gel to nitrocellulose membrane (Bio-Rad) by wet electroblotting apparatus (BioRad). The nitrocellulose membranes were then blocked in PBS containing 5% w/v nonfat dry milk and further incubated with the indicated primary antibodies and secondary antibodies for western blot analysis. Primary antibodies used included anti-GST (#2622; Cell Signaling Technology), anti-phospho-H3 (Ser10) (#06–570; Millipore), anti-Histone H3 (#ab1791; Abcam), anti-phospho-MAPK (#9106; Cell Signaling Technology), anti-β-Tubulin (sc-58884; Santa Cruz), and anti-Cyclin B1 (#AP11096c, Abgent). Secondary antibodies used were IRDye-680RD conjugated anti-mouse-IgG (#925–68070; Li-Cor) and IRDye-800CW-conjugated anti-rabbit-IgG (#925–32211; Li-Cor). Membranes were scanned using a Li-Cor Odyssey CLx Imager. Band intensities were normalized to loading controls (total H3 or β-Tubulin levels) and quantified in ImageJ.

Bisht J., LeValley P., Noren B., McBride R., Kharkar P., Kloxin A., Gatlin J, & Oakey J. (2019). Light-Inducible Activation of Cell Cycle Progression in Xenopus Egg Extracts Under Microfluidic Confinement. Lab on a chip, 19(20), 3499-3511.

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Immunofluorescence Microscopy of C. elegans

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A Zeiss motorized Axioplan 2 microscope with a 63× PlanApo (numerical aperture 1.4) objective lens was employed for fluorescence microscopy. Images were acquired with an AxioCam MRm camera and AxioVision acquisition software. Some images were acquired using an apotome adaptor (Zeiss), as indicated.
Anti-INX antibodies, anti-GFP monoclonal antibody 3E6, and anti-phospho-H3 (Ser10; Millipore) antibodies were used to stain dissected gonads (fixed in 3% formaldehyde in MRWB buffer for 1.25 hr, followed by a 20-min incubation in 10 mM DTT in Tris–Triton buffer; Finney and Ruvkun 1990 (link)), or whole-animal mounts (fixed in 1% formaldehyde for 1.5 hr; Finney and Ruvkun 1990 (link)). Fixed specimens were blocked overnight (5% BSA, 1× PBS, 0.5% Tween-20, 0.02% sodium azide) and stained with the appropriate primary or secondary antibodies in the same solution. Anti-dpMPK-1 staining followed Lee et al. (2007) (link).
DAPI staining of live embryos was carried out by dissecting gravid adults in a 0.25μg/ml DAPI solution. Embryos released by dissection were incubated for ∼10 min, washed twice, and mounted for examination.

Starich T.A., Hall D.H, & Greenstein D. (2014). Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans. Genetics, 198(3), 1127-1153.

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