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Ab2832

Manufactured by Abcam
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Ab2832 is a monoclonal antibody that recognizes the caspase-3 protein. Caspase-3 is a key enzyme involved in the execution phase of cell apoptosis. This antibody can be used to detect and quantify the expression of caspase-3 in various biological samples.

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Lab products found in correlation

Ab4729, by Abcam (6 mentions) Ab14984, by Abcam (3 mentions) Ez chip chromatin immunoprecipitation kit, by Merck Group (3 mentions) Pan acetyl h4, by Merck Group (2 mentions) Pcr purification kit, by Qiagen (2 mentions) Bioruptor sonicator, by Diagenode (2 mentions) Goat anti rabbit igg, by Cell Signaling Technology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Apotome module, by Zeiss (1 mentions) Alexa fluor 594 red, by Thermo Fisher Scientific (1 mentions) Ab7817, by Santa Cruz Biotechnology (1 mentions) Harmony software, by PerkinElmer (1 mentions) Bp1600, by Merck Group (1 mentions) Operetta, by PerkinElmer (1 mentions) Ab1791, by Abcam (1 mentions) Ab31419, by Abcam (1 mentions) Td buffer, by Illumina (1 mentions) Thruplex dna seq kit, by Takara Bio (1 mentions) Tn5 transposes, by Illumina (1 mentions) Hiseq x ten, by Illumina (1 mentions)

16 protocols using ab2832

1

Chromatin Immunoprecipitation with Enzymatic Digestion

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Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP®
Enzymatic Chromatin IP Kit (#9003, CST, U.S.A.) according to the
manufacturer’s instruction. Cells were harvest at 80–90%
confluence after siRNA transfection. Briefly, cells were cross-linked with
1% formaldehyde for 15 min, and then terminated with glycine before
scrapped from culture dishes. Afterwards, the collected cells were digested with
nuclease to break the cross-linked chromatin to 100–200 bp length. Then
appropriate amount of chromatin was immunoprecipitated using anti-CBP (ab2832,
Abcam, U.S.A.), anti-P300 (ab14984, Abcam, U.S.A), anti-histone H3A (acetyl K27)
(ab4729, Abcam, U.S.A.), and anti-histone H3A (acetyl K18) (Cat.#17-10111,
Millipore, U.S.A.); goat anti-Rabbit IgG and goat anti-Mouse IgG were used as
negative control respectively. The immunoprecipitation reaction was performed
overnight at 4°C with rotation. The next day, the precipitated chromatin
were washed and eluted from the antibody/protein G magnetic beads, and then DNA
purification was performed and analyzed by qPCR with specific primers listed in
Table 1.
Liang Y., Wu Y., Chen X., Zhang S., Wang K., Guan X., Yang K., Li J, & Bai Y. (2017). A novel long noncoding RNA linc00460 up-regulated by CBP/P300 promotes carcinogenesis in esophageal squamous cell carcinoma. Bioscience Reports, 37(5), BSR20171019.
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2

Visualizing Phospho-Tau in Tissue Sections

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For heat‐induced epitope retrieval, floating sections were kept in sodium citrate (pH 6, 10 mM, 37°C, 30 min). After permeabilization (PBS1X/Triton X‐100 2%, 15 min), unspecific labeling was blocked (PBS1X/Triton X‐100 0.1%/horse serum 5%, 1 h at RT) and slices were incubated overnight with polyclonal anti‐CBP antibody (1/50; ab2832; Abcam), washed, and further incubated with anti‐rabbit Alexa Fluor 594 (red, 1/1,000) antibody (Invitrogen, Thermo Fisher Scientific) for 1 h. IHC was performed sequentially and the second antibody was added in a second step. Slices were incubated overnight with monoclonal anti phospho‐Tau (Thr212, Ser214) antibody (1/1,000; AT100, MN1060 Thermo Fisher Scientific), washed, and further incubated with anti‐mouse Alexa Fluor 488 (green, 1/1,000) antibody (Invitrogen, Thermo Fisher Scientific). Slices were incubated with the Hoechst dye 33342 (1 mg/ml; 5 min) and mounted in Mowiol for observation. Acquisitions were performed using a fluorescence microscope coupled with an ApoTome module (Zeiss). Photomicrographs were captured using the z‐stack mode with 0.5 μm of thickness between slices, 18 in‐depth slices were taken and flattened using the maximum intensity projection (MIP).
Chatterjee S., Cassel R., Schneider‐Anthony A., Merienne K., Cosquer B., Tzeplaeff L., Halder Sinha S., Kumar M., Chaturbedy P., Eswaramoorthy M., Le Gras S., Keime C., Bousiges O., Dutar P., Petsophonsakul P., Rampon C., Cassel J., Buée L., Blum D., Kundu T.K, & Boutillier A. (2018). Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator. EMBO Molecular Medicine, 10(11), e8587.
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3

Chromatin Immunoprecipitation Profiling of Osteoclast Precursors

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ChIP was performed as previously described 14 (link). Briefly, 2 × 107 human primary OCPs were fixed by adding formaldehyde directly to the medium to a final concentration of 1 % for 5 min. Cells were harvested, washed, and lysed. Chromatin was sheared by sonication to a length of approximately 500 base pairs using a Bioruptor sonicator (Diagenode). Sheared chromatin was precleared and then immunoprecipitated with antibodies; c-myc (Abcam; ab56), Brd4 (Bethyl Lab; A301-985A), pan acetyl-H4 (Millipore; 06-866), and CBP (Abcam; ab2832). Immune complexes were subsequently collected and washed, and DNA crosslinking was reversed by heating at 65°C overnight. Following proteinase K digestion, DNA was recovered using the PCR purification kit (Qiagen) and real time PCR was performed to detect the occupancy of target proteins. Signals obtained from the ChIP are divided by signals obtained from an input sample. This input sample represents the amount of chromatin used in the ChIP. The primer sequences are listed in Supplementary Table 3.
Park-Min K.H., Lim E., Lee M.J., Park S.H., Giannopoulos E., Yarilina A., van der Meulen M., Zhao B., Smithers N., Witherington J., Lee K., Tak P.P., Prinjha R.K, & Ivashkiv L.B. (2014). Inhibition of Osteoclastogenesis and Inflammatory Bone Resorption by Targeting BET Proteins and Epigenetic Regulation. Nature communications, 5, 5418.
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4

ChIP-Seq protocol for histone acetylation

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ChIP was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (#9003, CST, USA). First, cells were harvested with protein cross linked to DNA using 1% formaldehyde. Then, chromatin was digested to lengths of ~100–900 bp using Micrococcal Nuclease. Next, 5–10 μg of digested and cross-linked chromatin combined with 2 μg of antibody was used per immunoprecipitation. Anti-KAT3A/CBP (ab2832, Abcam) and anti-histone H3 (acetyl K27) (ab4729, Abcam) were employed in ChIP assay, with goat anti-rabbit IgG used as a negative control. The retrieved DNA was quantified by real-time qPCR analysis with the specific primers listed in Supplementary Table 1. The results were calculated as a percentage of input DNA using the same formula as for the RNA pull-down.
Liang Y., Chen X., Wu Y., Li J., Zhang S., Wang K., Guan X., Yang K, & Bai Y. (2018). LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein. Cell Death and Differentiation, 25(11), 1980-1995.
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5

Immunofluorescence Analysis of Cellular Proteins

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Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences 15710) for 20 min and permeabilized in 0.01% Triton X100 for 1 h at room temperature. Cells were blocked in 5% BSA (Sigma BP1600) for 1 h, and primary antibodies added and incubated at 4°C overnight. Primary antibodies used are as follows in 5% BSA: aSMA (abcam ab7817 1:1000), YAP (Santa Cruz Biotechnology sc‐101199, 1:500), Methylated Lysine (Novus Biologics NB600‐824, 1:500), Acetylated Lysine (abcam ab190479, 1:300), Histone H3 acetylation (EMD Millapore 06‐599, 1:1000), and CREBBP (abcam ab2832, 1:1000). Cells were washed, and secondary antibodies were added for 30 min. Cells were imaged with automated Perkin Elmer Operetta. Cell area, nuclear area, antibody intensity was quantified using Perkin Elmer's Harmony software. For αSMA measurements, values were normalized to Cellmask intensity.
Walker C.J., Batan D., Bishop C.T., Ramirez D., Aguado B.A., Schroeder M.E., Crocini C., Schwisow J., Moulton K., Macdougall L., Weiss R.M., Allen M.A., Dowell R., Leinwand L.A, & Anseth K.S. (2022). Extracellular matrix stiffness controls cardiac valve myofibroblast activation through epigenetic remodeling. Bioengineering & Translational Medicine, 7(3), e10394.
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6

RNA Immunoprecipitation Assay for Protein-RNA Interactions

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RIP assay was performed using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Cat. #17-701, Millipore, USA). Cells were cultured in 75-cm2 flasks to 80–90% confluence. Then, cells were harvested by scraping. One RIP reaction required 100 μl of cell lysate from ~2.0 × 107 cells. Next, 5 μg of purified antibodies and corresponding IgG was added to the 100-μl cell lysate, and the mixture was incubated with rotation overnight at 4 °C. Anti-SNRNP70 (Cat. #CS203216, Millipore), anti-KAT3A/CBP (ab2832, Abcam) and normal rabbit IgG (Cat. #PP64B, Millipore) were used for RIP assay. The immunoprecipitated RNA was purified and analyzed with RT-qPCR, and the results were calculated as a percentage of input RNA using the following formula: Percent input = 2% × 2 (C[T] 2% input sample − C[T] IP sample).
Liang Y., Chen X., Wu Y., Li J., Zhang S., Wang K., Guan X., Yang K, & Bai Y. (2018). LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein. Cell Death and Differentiation, 25(11), 1980-1995.
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7

Quantification of CREBBP Protein Levels

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SKK-1 cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein quantification was done using the BCA Protein Assay Kit (Thermo Scientific) and after boiling in Laemli buffer, equal protein concentrations were loaded and separated on SDS-polyacrylamide gel electrophoresis prior to transfer overnight to nitrocellulose membranes. After blocking with non-fat milk, membranes were incubated with primary antibodies and then with appropriate secondary fluorophore-conjugated secondary antibodies (LI-COR Biosciences, IRDye, 1 : 20,000). The dried membranes were scanned with an Odyssey CLx Imager. Protein levels were quantified using the Odyssey and the band density normalized using the density of the histone H3 signal. The following antibodies were used: anti-CREBBP (Abcam, Cambridge, UK, ab2832, 1 : 1000 dilution) and anti-histone H3 (Abcam, ab1791, 1 : 10,000 dilution).
Diesch J., Le Pannérer M.M., Winkler R., Casquero R., Muhar M., van der Garde M., Maher M., Herráez C.M., Bech-Serra J.J., Fellner M., Rathert P., Brooks N., Zamora L., Gentilella A., de la Torre C., Zuber J., Götze K.S, & Buschbeck M. (2021). Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis. Nature Communications, 12, 6060.
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8

ChIP-seq and ATAC-seq Analysis of Transcription Factors

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ChIP-seq and ATAC-seq were performed as described previously (Ohba et al., 2015 (link)). Briefly, chromatin prepared from 1 × 107 PANC-1 cells was immunoprecipitated with anti-β-catenin (Cell signaling, D10A8), CBP (Abcam, ab2832), p300 (Abcam, ab14984), and c-JUN (Abcam, ab31419) antibodies. Libraries were prepared with ThruPLEX DNA-seq Kit (Takara-bio, R400674). For ATAC-seq, 50,000 nuclei were labeled with TD buffer (Illumina, 15027866) and Tn5 transposes (Illumina, 15027865). Libraries were sequenced with Illumina Hiseq X ten. Sequence reads were mapped to the human genome (hg19) with bowtie (Langmead et al., 2009 (link)). Binding peaks were determined with the CisGenome peak caller (Ji et al., 2008 (link)). Common peaks were determined by intersection with bedtools. De novo motif analysis was performed using the Gibbs motif sampler provided in the CisGenome package (Ji et al., 2008).
Doi T., Hojo H., Ohba S., Obayashi K., Endo M., Ishizaki T., Katoh A, & Kouji H. (2022). Involvement of activator protein-1 family members in β-catenin and p300 association on the genome of PANC-1 cells. Heliyon, 8(2), e08890.
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9

Western Blot Analysis of Signaling Proteins

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Radio immunoprecipitation assay lysis buffer (Sigma‐Aldrich) was employed for protein extraction. Then, the proteins were separated by SDS‐PAGE for 2 hours and were transferred onto a PVDF membrane, which was then soaked in TBST containing 5% skim milk at 37°C for 1 hour. The primary antibodies rabbit anti‐PPP1R1B (#ab40801, 1:1000; Abcam, Shanghai, China), rabbit anti‐CREB (#ab32515, 1:1000; Abcam), rabbit anti‐CBP (#ab2832, 1 μg/mL; Abcam), and rabbit anti‐β‐Actin (#ab8227, 1:1000; Abcam) were added to the membranes, and the membranes were then incubated at 4°C overnight. After being using TBST to wash three times the membranes were subsequently incubated for 2 hours at 37°C, with goat anti‐rabbit IgG H&L secondary antibody (1:2000). The immunological reaction was detected with ECL Plus reagent (Amersham, Piscataway, NJ, USA). ImageJ 1.8.0 software was conducted to analyse data. β‐actin was employed as an internal reference protein.
Xian D, & Zhao Y. (2019). LncRNA KCNQ1OT1 enhanced the methotrexate resistance of colorectal cancer cells by regulating miR‐760/PPP1R1B via the cAMP signalling pathway. Journal of Cellular and Molecular Medicine, 23(6), 3808-3823.
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10

ChIP and RIP Assays for Histone Modifications

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ChIP was performed using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Burlington, MA) according to the manufacturer’s protocol. Briefly, cross-linked chromatin was sonicated into 200–1000 bp fragments. The chromatin was immunoprecipitated using anti-H3K27ac (Abcam, ab4729, Cambridge, MA)and anti-CBP antibodies (Abcam, ab2832). Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used for RIP and anti-CBP antibody was used to pull down AGAP2-AS1.
Dong H., Wang W., Mo S., Chen R., Zou K., Han J., Zhang F, & Hu J. (2018). SP1-induced lncRNA AGAP2-AS1 expression promotes chemoresistance of breast cancer by epigenetic regulation of MyD88. Journal of Experimental & Clinical Cancer Research : CR, 37, 202.
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