After transfer process, the primary antibodies against CHOP purchased from Cell Signaling Technology; Danvers, MA, USA, Bcl-2, cytochrome c, Snail1, Zeb1, and histone H2B purchased from Abcam; and PKC, MEK, p-ERK, ERK, and β-actin purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA were maintained overnight at 4 °C. As detailed in previous research [70 (link)].
Histone h2b
Manufactured by Abcam
Sourced in United States
Histone H2B is a core histone protein that is a component of nucleosomes in eukaryotic cells. Histones play a fundamental role in the organization and compaction of DNA within the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Cytochrome c, by Abcam (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Abcam (3 mentions)
Caspase 3, by Santa Cruz Biotechnology (2 mentions)
Snail1, by Abcam (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Lc3a b antibody, by Cell Signaling Technology (1 mentions)
X omat ar film, by Kodak (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
H3k4me2, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
H3k4me1, by Cell Signaling Technology (1 mentions)
H3k9 14ac, by Cell Signaling Technology (1 mentions)
H3k18ac, by Cell Signaling Technology (1 mentions)
H3k4me3, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
11 protocols using histone h2b
1
Protein Expression Analysis Protocol
2
Immunofluorescence Analysis of Autophagy and Transcription Factors
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Cells were seeded at a density of 5 × 105 cells per well in six well plates containing cover glasses/slips in each well. Following indicated transfections, serum starvation and time points, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) and then permeabilized in pre-chilled 100% methanol for 10 minutes at −20 °C. Cells were then washed with 1x PBS for 5 minutes, blocked and incubated overnight at 4 °C with antibodies raised against LC3A/B antibody (Cell Signaling Technology), FLAG tag (Cell Signaling Technology), NRF2 (Novus Biologicals) and Histone H2B (Abcam). Conjugated secondary antibodies used include goat anti-mouse and anti-rabbit IgG (H + L) Fluorescein (FITC) (Jackson ImmunoResearch Labs) as well as anti-rabbit and anti-mouse IgG Alexa Fluor® 555 conjugates (Cell Signaling Technology). Fixed cells were incubated with diluted secondary antibodies for 1 hour at room temperature after which they were washed three times with 1x PBS and mounted with ProLong Gold Antifade Reagent (Cell Signaling Technology). Analysis was carried out in an epifluorescence microscope using single interference filters set for green (fluorescein isothiocyanate, FITC), red (Texas Red), and blue (DAPI) as well as dual (red/green) band pass filters.
3
Protein Expression Analysis of ER Stress Markers
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Primary antibodies against CHOP (C/EBP homologous protein, marker of ER stress) purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA); Bcl-2, cytochrome c, and histone H2B purchased from Abcam (Abcam, Cambridge, UK); and caspase 3 and β-actin purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA) were maintained overnight at 4 °C. Blots were developed with ECL reagents (Pierce) and exposed using Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY, USA) for 3–5 min.
4
Immunoblotting of Apoptosis-Related Proteins
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Primary antibodies against GRP 78 and CHOP bought from Cell Signaling Technology (Danvers, MA, USA), p21, Bcl 2, cytochrome c, and histone H2B (bought from Abcam), and cyclin D1, caspase 3, and β-actin (bought from Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Further protocol details are recounted in our previous article [62 (link)].
5
Quantifying Neutrophil Extracellular Traps
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Neutrophil extracellular traps in the lung sections were quantified by triple immunolabelling, as described previously (7 (link)). Briefly, lung sections (5 μm) were stained with antibodies against histone H2B (Abcam) and myeloperoxidase (MPO, Abcam), and DAPI (Life Technologies). NETs were identified as single strands or clusters, and scored according to pre-determined criteria (0–10). Twenty fields were analyzed, and the sum was calculated for the final NETs score.
6
Western Blot Histone Modification Analysis
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Non-specific binding to nitrocellulose membranes was blocked in TBS-Tw (Tris-buffered saline with 0.05% Tw-100) and 5% (w/v) semi-skim powdered milk (Sainsbury's) for 30 min at room temperature (RT). Membranes were then washed 3× for 5 min with TBS-Tw. The appropriate dilution of primary antibodies was prepared in TBS-Tw and 5% (w/v) BSA and membranes were blotted with primary antibodies overnight at 4°C. Following overnight incubation membranes were washed three times for 5 min with TBS-Tw, the membranes were then incubated in the appropriate HRP-linked secondary antibody for 1 h in TBS-Tw and 5% (w/v) semi-skim powdered milk at RT. Finally, the membranes were washed 6× for 5 min with TBS-Tx followed by two rinses in TBS. Immune-complexes were detected by enhanced chemiluminescence (ECL, Amersham Biosciences).
Primary antibodies and dilutions used in immunoblotting experiments were as follows: ubH2B @ 1:250 (Medimabs), H3K4me3 @ 1:1000 (Cell Signalling), H3K4me2 @ 1:1000 (Cell Signalling), H3K4me1 @ 1:1000 (Cell Signalling), H3K9/14ac @ 1:1000 (Cell Signalling), H3K18ac @ 1:500 (Cell Signalling), histone H3 @ 1:1000 (Abcam), histone H2B @ 1:1000 (Abcam), α-tubulin @ 1:2000 (Sigma).
Primary antibodies and dilutions used in immunoblotting experiments were as follows: ubH2B @ 1:250 (Medimabs), H3K4me3 @ 1:1000 (Cell Signalling), H3K4me2 @ 1:1000 (Cell Signalling), H3K4me1 @ 1:1000 (Cell Signalling), H3K9/14ac @ 1:1000 (Cell Signalling), H3K18ac @ 1:500 (Cell Signalling), histone H3 @ 1:1000 (Abcam), histone H2B @ 1:1000 (Abcam), α-tubulin @ 1:2000 (Sigma).
7
Histone Protein Identification in Hair
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Hair proteins (50 μg) were separated on 12% polyacrylamide gels, transferred onto 0.45-μm PVDF membranes (BioRad, Hercules, CA), blocked and probed overnight at 4 °C with the primary antibodies. Antibodies against histone H1.0 (Abcam), histone H2A (Cell Signaling Technology), histone H2B (Abcam), histone H3 (Cell Signaling Technology), and histone H4 (Cell Signaling Technology), and secondary antibodies against rabbit immunoglobulins (Dako) were used in this study.
8
Western Blot Analysis of Nrf2, HO-1, and NF-κB Signaling
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Cell extracts were prepared in RIPA buffer (Beyotime, China). Nuclear extracts were prepared with a Nuclear Extract Kit (Thermo Scientific, USA) according to the manufacturer's recommendations. Then, Western blot analysis was performed according to standard procedures. Antibodies were used at the following concentrations: Nrf2 (Abcam), 1 : 1000; HO-1 (Santa Cruz), 1 : 500; c-jun (Santa Cruz), 1 : 1000; Histone H2B (Abcam), 1 : 1000; GAPDH (Santa Cruz), 1 : 1000; p65 (Santa Cruz), 1 : 1000; c-jun (Santa Cruz), 1 : 1000.
9
Western Blot Analysis of Cellular Proteins
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Cells were lysed in RIPA buffer (Vazyme, CHINA) for 45min on ice and centrifuged at 2500g for 5min at 4°C. After quantification using One Drop® OD-1000 Spectrophotometer (Nanjing Wuyi Corporation, CHINA), whole cell lysates were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore, USA). The membranes were blocked in 5% BSA (Sangon, CHINA) and then incubated with primary antibodies for β-ACTIN (Proteintech, USA), β-TUBULIN (Proteintech, USA), CA9 (Absolute Antibody, UK), FBL (Proteintech, USA), HIF1α (Novus, USA), HIF2α (BD, USA), and Histone H2B (Abcam, UK). The PVDF membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (Proteintech, USA). Immunoreactive proteins were visualized using the SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA) on ChemiDoc-It (UVP, UK).
10
Chromatin Enrichment Protocol for Proteomics
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In order to more specifically enrich for chromatin and reduce background from cellular contaminants and from the non-chromatin associated bait, we improved the stringency of our in-house ChIP protocol (3 (link)) by incorporating a few steps from the Chromatin enrichment for proteomics (CheP) protocol (32 (link)) during the preparation of our input material. subsequently we followed a traditional ChIP protocol in which we have enriched for the dCas9 bait and its associated complexes using an Anti-V5 Agarose Affinity Gel (Sigma). To further eliminate background originating from the purification of non-chromatin associated bait complexes we additionally added a histone enrichment step, downstream of the bait enrichment step, using histone H2B (Abcam) and H3 antibodies (Abcam) conjugated to Protein G Sepharose™ 4 Fast flow (GE Healthcare) and Protein A Sepharose™ 4 Fast flow (GE Healthcare) beads by dimethyl pimelimidate cross-linking as described in (33 (link)).
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