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Anti cenp a

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Anti-CENP-A is a primary antibody that specifically binds to the CENP-A (Centromere Protein A) protein. CENP-A is a histone H3 variant that is a essential component of the centromeric chromatin.

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Lab products found in correlation

Anti gapdh, by Cell Signaling Technology (3 mentions) Pvdf membrane, by GE Healthcare (2 mentions) Tgx gel, by Bio-Rad (2 mentions) Anti vinculin, by Merck Group (2 mentions) Trans blot turbo transfer system, by Bio-Rad (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Anti histone h3, by Merck Group (1 mentions) Anti phospho histone h3 ser10, by Cell Signaling Technology (1 mentions) Anti lig4, by GeneTex (1 mentions) Anti dna pkcs, by Fortis Life Sciences (1 mentions) Anti lig3, by Fortis Life Sciences (1 mentions) Anti brca2, by Fortis Life Sciences (1 mentions) Anti parp, by BD (1 mentions) Hrp conjugated secondary antibody, by GE Healthcare (1 mentions) H3k27me3, by Cell Signaling Technology (1 mentions) H3k9me3, by Abcam (1 mentions) Anti tubulin, by Cell Signaling Technology (1 mentions) Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594, by Thermo Fisher Scientific (1 mentions) Anti h3k9me3, by Abcam (1 mentions)

8 protocols using anti cenp a

1

Analysis of CENP-A Levels in Cell Lines

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Whole cell lysates were collected from the different cell lines, treated or not with 500 μM IAA (I5148, Sigma) dissolved in ddH2O. For DLD-1 cells, proteins were separated by 15% SDS-PAGE and transferred onto PVDF membranes (GE healthcare). Immunoblot analysis was performed by blocking the membrane with 5% skim milk and probing with anti-ACA (Antibodies Incorporated, 2 μg/mL) and anti-α-tubulin (Clone DM1A, 1:5000) antibodies. Three independent gels were probed to quantify CENP-A-T2A levels using Fiji software (Schindelin et al., 2012 (link)). Proteins from RPE-1 whole cell lysates were separated by SDS-PAGE using 12% TGX gels (BioRad) or hand casted 15% gels, and transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (BioRad). Immunoblot analysis was performed by blocking the membrane with 5% skim milk and probing overnight at 4°C with anti-CENP-A (1:1000, Cell Signaling #2186), anti-Vinculin (1:2000, Sigma #V9264) and anti-GAPDH (1:5000, Cell Signaling #2118) antibodies.
Salinas-Luypaert C., Allu P.K., Logsdon G.A., Dawicki-McKenna J.M., Gambogi C.W., Fachinetti D, & Black B.E. (2021). Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub. Cell reports, 37(5), 109924.
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2

Analyzing DNA Damage Response Proteins

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Whole-cell extracts were collected in SDS sample buffer and boiled for 10 mins. Samples were resolved by SDS-PAGE, transferred to PVDF, and blocked with 5% milk in PBST (PBS, 0.1% Tween-20). The following primary antibodies were used at 1:1,000 dilution (unless noted) in PBST: anti-CENP-A (Cell Signaling, 2186), anti-phospho histone H2AX (ser139) clone JBW301 (EMD Millipore, 05-636), anti-phospho histone H3 (ser10) (Cell Signaling, 9706), 1:4,000 anti-histone H3 (Sigma H0164), anti-LIG4 (GeneTex, GTX100100), anti-DNA-PKcs (Bethyl, A300-516A), anti-LIG3 (Bethyl, A301-637A), anti-PARP (BD Pharmingen, 556362, kindly provided by X. Wu, The Scripps Research Institute, USA), anti-BRCA2 (Bethyl, A303-434A), anti-RAD51 (Abgent, AM8421b), and 1:2,000 anti-GAPDH (Cell Signaling, 14C10). Blots were probed with 1:4,000 dilutions of HRP-conjugated secondary antibodies (GE Healthcare) and exposed to film. All unprocessed film scans with the appropriate size markers are provided in Supplementary Fig. 6.
Ly P., Teitz L.S., Kim D.H., Shoshani O., Skaletsky H., Fachinetti D., Page D.C, & Cleveland D.W. (2016). Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by canonical NHEJ. Nature cell biology, 19(1), 68-75.
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3

Native ChIP for Liver Cell Histones

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Native ChIP was performed using the protocol described previously [29 (link)] on homogenized liver cells using anti-CENP-A (Cell Signaling Technologies, Cat # C51A7), H3K9me3 (Abcam, Cat # ab8898), H3K27me3 (Cell Signaling Technologies, Cat # C36B11) and IgG (Abcam, Cat # ab46540) antibodies using the following modification. After MNase digestion and needle extraction, low (150 mM) and high (350 mM) NaCl containing fractions were combined for antibody binding and downstream steps.
Packiaraj J, & Thakur J. (2023). DNA satellite and chromatin organization at house mouse centromeres and pericentromeres. bioRxiv.
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4

Immunofluorescence Staining of Cell Markers

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Cells were grown on glass coverslips in 6 well dishes. Media was removed, cells were washed twice with PBS, and then treated with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were washed 3 times in PBS and treated with a quenching solution (75 mM NH4Cl and 20 mM Glycine in PBS) for 10 min. Fixed coverslips then permeabilized with PBS-Triton X-100 0.1% for 2 min on ice. Blocking and antibody dilutions were performed with 5% BSA. The primary antibody was diluted at a ratio of 1:500 and applied for 1 h at room temperature. The secondary antibody was diluted as direction and applied for 45 min at room temperature. Prolong Gold with DAPI (ThermoFisher P36931) was used for mounting. The primary antibodies used were anti-CENPA (Cell Signaling 2047), anti-tubulin (Cell Signaling 2146) and anti-H3K9me3 (Abcam ab176916), and the secondary antibodies used were donkey anti-rabbit Alexa Fluor 488 (Thermo A-21206) and goat anti-rabbit Alexa Fluor 594 (Thermo A-11012).
Gutbrod M.J., Roche B., Steinberg J.I., Lakhani A.A., Chang K., Schorn A.J, & Martienssen R.A. (2022). Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4. Nature Communications, 13, 1001.
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5

Analysis of CENP-A Levels in Cell Lines

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Whole cell lysates were collected from the different cell lines, treated or not with 500 μM IAA (I5148, Sigma) dissolved in ddH2O. For DLD-1 cells, proteins were separated by 15% SDS-PAGE and transferred onto PVDF membranes (GE healthcare). Immunoblot analysis was performed by blocking the membrane with 5% skim milk and probing with anti-ACA (Antibodies Incorporated, 2 μg/mL) and anti-α-tubulin (Clone DM1A, 1:5000) antibodies. Three independent gels were probed to quantify CENP-A-T2A levels using Fiji software (Schindelin et al., 2012 (link)). Proteins from RPE-1 whole cell lysates were separated by SDS-PAGE using 12% TGX gels (BioRad) or hand casted 15% gels, and transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (BioRad). Immunoblot analysis was performed by blocking the membrane with 5% skim milk and probing overnight at 4°C with anti-CENP-A (1:1000, Cell Signaling #2186), anti-Vinculin (1:2000, Sigma #V9264) and anti-GAPDH (1:5000, Cell Signaling #2118) antibodies.
Salinas-Luypaert C., Allu P.K., Logsdon G.A., Dawicki-McKenna J.M., Gambogi C.W., Fachinetti D, & Black B.E. (2021). Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub. Cell reports, 37(5), 109924.
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6

Immunoblot Protocol for Protein Analysis

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For immunoblot, normalized cell lysates or immunoprecipitation samples were separated on SDS-PAGE gels and transferred on nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). The blots were probed with the following primary antibodies; anti-Flag (Sigma, St Louis, MO), anti-HA (Covance, Princeton, NJ), anti-H3 (Cell signaling, Danvers, MA), anti-p-H3S10 (Abcam, Cambridge, UK), anti-Aurora B (Abcam, Cambridge, UK), anti-PLK1 (Santa Cruz, Dallas, TX), anti-ACA (Antibodies Incorporated, Davis, CA), anti-CENP-A (Cell Signaling, Danvers, MA), anti-phospho serine (Sigma, St Louis, MO). Phosphorylation-specific antibody for Mis18α was generated by injecting synthetic phospho-peptide to rabbits and purified using phospho-peptide affinity chromatography (AbClone, Seoul, South Korea). Peptide sequence used for injection is as follows; 5′-CESPLLEKRL(pS)EDSSR-3′.
Lee M., Kim I.S., Park K.C., Kim J.S., Baek S.H, & Kim K.I. (2017). Mitosis-specific phosphorylation of Mis18α by Aurora B kinase enhances kinetochore recruitment of polo-like kinase 1. Oncotarget, 9(2), 1563-1576.
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7

Comprehensive Antibody Validation Protocol

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The following antibodies were used: LACC1 E-12 (anti-FAMIN; sc-376231), E-7 (sc-374553), H-6 (sc-376064) from Santa Cruz; anti-β actin (ab6276) and anti-PMP70 (ab3421) from Abcam; anti-β-tubulin (2128), anti-calnexin (2679), anti-calreticulin (12238), anti-catalase (12980), anti-caveolin-2 (8522), anti-CENP-A (2186), anti-COX-IV (4850), anti-EEA1 (3288), anti-fibrillarin (2639), anti-histoneH3 (4499), anti-LAMP1 (9091), anti-LC3B (2868), anti-NUP98 (2598), anti-PDI (2446), anti-Rab5 (3547) and anti-syntaxin-6 (2869) from Cell Signaling; anti-ABCD3 (HPA032027) from Sigma Aldrich.
Assadi G., Vesterlund L., Bonfiglio F., Mazzurana L., Cordeddu L., Schepis D., Mjösberg J., Ruhrmann S., Fabbri A., Vukojevic V., Percipalle P., Salomons F.A., Laurencikiene J., Törkvist L., Halfvarson J, & D’Amato M. (2016). Functional Analyses of the Crohn’s Disease Risk Gene LACC1. PLoS ONE, 11(12), e0168276.
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8

Immunofluorescence and Western Blot Protocols

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Monoclonal mouse antibody against α-tubulin B-5-1-2 (T5168, Merck, Darmstadt, Germany) was used for immunofluorescence analysis (IF) and Western blotting (WB) at 1:2000. Polyclonal rabbit antibodies against the following proteins were used: anti-Kid (AKIN12, Cytoskeleton Inc., Denver, CO, USA) for WB at 1:2000; anti-KIF4A (A301-074A, BETHYL, Montgomery, TX, USA) for WB at 1:2000, and anti-CENP-A (#2186, Cell Signaling Technology, Danvers, MA, USA) for IF at 1:400. Human auto-antiserum against centromeric antigen (ACA; IF 1:30,000) was a gift from T. Hirota.
Kuniyasu K., Iemura K, & Tanaka K. (2018). Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome Missegregation in Cancer Cell Lines. Biomolecules, 9(1), 10.
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