Total protein from cultured cells was extracted in a T-PERbuffer (Thermo) and Halt protease and Phosphatase Inhibitor Cocktail (Thermo). Total protein was quantified using a BCA protein assay kit (Thermo). Proteins and ladder (BIO-RAD) were run on 4–20% Mini-ProteanTGX Gels from BIO-RAD. Membranes were washed with buffered Tris. Anti-V5 conjugated to horseradish peroxidase antibody (diluted 1:4000, Invitrogen) was used to detect BirA. Anti-Histone H3 antibody (Abcam) was used at a 1:4000 dilution and an anti-rabbit conjugated to horseradish peroxidase was used as a secondary antibody (diluted 1:20000, Thermo). All membranes were blocked and antibodies diluted in a 1% BSA/TBS-Tween solution. Membranes were developed with the SuperSignal West Pico chemiluminescent substrate (Thermo) and stripped with Restore western blot stripping buffer (Thermo).
Anti histone h3 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-histone H3 antibody is a primary antibody that specifically binds to histone H3, a core component of nucleosomes in eukaryotic cells. This antibody can be used in various applications, such as western blotting, immunoprecipitation, and chromatin immunoprecipitation, to study histone H3 and its post-translational modifications.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Cell Signaling Technology (3 mentions)
Western lighting chemiluminescence reagent plus, by PerkinElmer (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Axio imager a1 inverted fluorescence microscope, by Zeiss (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Ampk and acc antibody sampler kit, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Ab1791, by Abcam (2 mentions)
Anti acetyl histone h4, by Merck Group (2 mentions)
Anti erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti α tubulin antibody, by Merck Group (2 mentions)
Anti parp antibody, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
55 protocols using anti histone h3 antibody
1
Western Blot Analysis of Protein Extracts
2
Quantification of Citrullinated Histone 3 in Septic Mice
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Citrullinated histone 3 protein was determined in neutrophils isolated from the blood of mice 24 h after CLP induction. Proteins (20 μg per lane) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 15% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in 0.05% PBS-Tween containing 5% milk (Bio-Rad Laboratories) and incubated with the anti-histone H3 antibody (citrulline R2 + R8 + R17, 1:1,000, Abcam) at 4 °C overnight. The primary antibody was detected by incubation with horseradish peroxidase-coupled second antibody (1:3,000 in PBS-Tween with 5% milk) at room temperature for 1 h. Chemiluminescence was detected by using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer LAS, Inc., Boston, MA). Films were developed using a standard photographic procedure and quantitative analysis of detected bands was carried out by densitometer scanning using VersaDoc Imaging System (BioRad Laboratories). Total protein was used as a loading control.
3
Comprehensive Protein Expression Analysis
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The anti-PARP, Survivin, Mcl-1, XIAP, Bcl-2, Bcl-XL, p15, p21, Cyclin D1, p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-JNK, JNK, p65, Tubulin, RelB, Caspases-7 and -8, cleaved Caspase-7 and -8 antibodies were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA). The anti-Cyclin B1 antibody was purchased from Abnova (Abnova, Taipei, Taiwan). The anti-Cyclin E1 antibody was purchased from Zymed (Zymed, San Francisco, CA). The anti-p27 antibody was purchased from Becton Dickinson (BD, San Diego, CA). The anti-A20 antibody was purchased from eBioscience (eBioscience, San Diego, CA). The anti-histone H3 antibody was purchased from Abcam (Abcam, Cambridge, UK). The anti-Caspase-3 and cleaved Caspase-3 antibodies were purchased from Imagenex (Imagenes, San Diego, CA). The anti-β-actin antibody was purchased from Sigma (Sigma, St. Louis, MO).
The MDA-MB-468 and SW527 cell lysate was subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with diluted primary antibodies, horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA), and Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and were visualized on an ImageQuant LAS4000 Biomolecular imager to determine the expression levels of specific proteins.
The MDA-MB-468 and SW527 cell lysate was subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with diluted primary antibodies, horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA), and Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and were visualized on an ImageQuant LAS4000 Biomolecular imager to determine the expression levels of specific proteins.
4
Protein Expression and Modification Analysis
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Western blotting was performed as described previously [30 (link)]. The anti-METTL3 antibody (#ab195352) and anti-Ki67 (#ab156956) were purchased from Abcam. The anti-IFIT2 antibody (#DF8962) was purchased from Affbiotech. The anti-H3K4me3 antibody (#39160) was purchased from Active Motif. The anti-β-actin antibody (#4970) and anti-rabbit IgG, HRP-linked antibody (#7071) was purchased from Cell Signaling Technology. The anti-m6A antibody (#202003) was purchased from Synaptic Systems. The anti-histone H3 antibody (#ab21054) was purchased from Abcam. The anti-YTHDF2 antibody (#24744-1-AP) was purchased from Proteintech.
5
Visualizing Neutrophil Extracellular Traps
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NETs were stained for immunofluorescence microscopy as described (32 (link)) using methodology modified from (33 (link)). In brief, 5 ×105 neutrophils were added to fibrinogen-coated coverslips, stimulated for 4 h with 40 nM PMA, 0.5 mM MnCl2 or varying concentrations of leukadherin-1 (LA-1; Sigma, UK), and fixed with 4% PFA. Coverslips were blocked and sequentially incubated with an anti-histone H3 antibody (Abcam, UK) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (Life Technologies, UK). Coverslips were washed, mounted, and sealed using with ProLong™ Gold antifade mountant with DAPI (Invitrogen, UK). Slides were visualized using a Zeiss Axio Imager.A1 inverted fluorescence microscope (Zeiss, Germany) and images analyzed using Image J.
6
Antibody Identification for Chromatin Immunoprecipitation
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Anti-dimethylated histone H3 lys36 antibody (MAB Institute, Inc.; #MABI0332-100), anti-trimethylated histone H3 lys36 antibody (MAB Institute, Inc.; #MABI0333-100), and anti-histone H3 antibody (Abcam; # ab1791) were purchased. The control antibody (Cell Signaling, normal rabbit IgG; #2729S) for ChIP assays was also purchased. The anti-KDM2A antibody produced in previous study was used28 (link). Anti-phosphorylated AMPKα antibody (Thr-172), anti-AMPKα antibody and β-actin antibody for immunoblotting were purchased (AMPK and ACC Antibody Sampler Kit, Cell Signaling; #9957 and Sigma, AC-15; #A5441).
7
Quantifying Neutrophil Histone H3 Expression
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Mouse bone marrow neutrophils were extracted via gradient centrifugation. Cell climbing tablets were placed in the 12-well plates, after which the extracted cells were added. After being cultured in a cell incubator at 37 °C and 5% CO2 for 30 min, rhCD177 was added, and then Phorbol 12-myristate 13-acetate (PMA) was added 30 min later. After 4 h, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS, and the cell membrane was broken using 0.25% TritonX-100. After another PBS wash, cells were inoculated on cell slides. The new slides were then incubated with PBS containing 5% goat serum for 1 h, and then with anti-histone H3 antibody (Abcam) overnight at 4 °C. One day later, slides were washed with PBS again and incubated at 25 °C for 2 h with Alexa Fluor 488 (green) labeled anti-rabbit IgG. Hoechst 33342 was used for nuclear staining after washing. Fluorescent immune samples were observed using a confocal microscope (Nikon A1R HD25, Tokyo, Japan) and analyzed using the NIS Elements AR 4.3 software.
8
Western Blot Analysis of BbElp3 Mutants
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All strains including wild type, ΔBbElp3, and ΔBbElp3::BbElp3 were inoculated in SDB liquid medium and grown with aeration for 3 d at 26 °C; then, the total protein was extracted and subjected to Western blotting as described [44 (link)]. Protein concentrations were quantified using the BCA Protein Assay Kit (KeyGen, Nanjing, China). Protein levels of β-actin and histone H3 were detected by use of an anti-β-actin antibody (Cell Signaling Technology, catalog #8457) and an anti-histone H3 antibody (Abcam, catalog #ab1791), respectively. The bulk acetylation of H3 was determined with H3Ac (H3 acetylation) antibody (Merck Millipore, catalog #06-599). After primary antibody binding, washing, and secondary antibody incubation using goat anti-rabbit IgG antibody (Boster, Wuhan, China), subsequent bands were detected by chemiluminescence examination (Amersham Biosciences, Shanghai, China). All experiments above were repeated three times. The intensities of all blots were quantified using ImageJ software (https://imagej.nih.gov/ij/ , accessed on 20 July 2017).
9
ChIP-qPCR Analysis of PPARγ Binding
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We used approximately one million HeLa cells per reaction. Chromatin shearing was performed using a sonicator (Branson). We carried out the ChIP using the ChIP Kit (Abcam, Cambridge, UK #ab500) following instructions of the manufacturer. For the pull-down step, the following antibodies and quantities were used: anti-Histone H3 antibody as positive control (Abcam, #ab1791; 2.5 μg), a Rabbit-anti-Mouse-Alexa Fluor 488 (ThermoFisher, Waltham, USA, #A-11,059; 5 μg), and an anti-PPARγ (Abcam, #ab59256; 5 μg). After DNA purification, we used 2 μl of DNA for qPCR, we carried out the reaction with the EDN1prom primers from Supplementary Table S3 using Power-Up SYBR Green Master Mix (ThermoFisher, Waltham, USA). For data analysis, we used the input percent method [100 × 2(InputCT−CT(IP)].
10
ChIP Assay for Transcription Factor Binding
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For Chromatin immunoprecipitation assay (ChIP) cells were cross-linked with a final concentration of 1% formaldehyde for 10 min at 37 °C, then washed and harvested in SDS lysis buffer (10% SDS; 0.5 M EDTA; 1 M Tris-HCl; containing proteinase inhibitor cocktail from Sigma-Aldrich, St. Louis, MO, USA) and sheared by sonication to fragment DNA. Samples were immunoprecipitated with 10 µg of anti-KLF6 antibody (polyclonal antibody KLF6 (R-173) or monoclonal antibody KLF6 (E-10) (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H3 antibody (Abcam, Cambridge, UK) or control IgG (Abcam) and protein-A/G agarose beads (Santa Cruz Biotechnologies). Following removal of cross-linked DNA/protein complexes by Proteinase K (Qiagen, Hilden, Germany) treatment, immunoprecipitated DNA was purified using QIAamp DNA Mini Kit (Qiagen) and used for PCR with ATG7 or BECLIN1 primers (Supplementary Table S3 ), encompassing the promoter region −200 bp to −400 bp upstream of transcriptional start site to amplify immunoprecipitated DNA, PCR products were visualized on an agarose gel.
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