Western blotting was performed as described previously [7 (link)]. The protein levels of Acetyl-Histone H3, Histone H3, Histone 2B, Bcl-2, and cleaved caspase-3 were analyzed with the following antibodies, respectively: Acetyl-Histone H3 (Millipore, Temecula, CA), Histone H3 (Millipore, Temecula, CA), Histone 2B (Santa Cruz, Santa Cruz, CA), Bcl-2 (Cell Signaling, Beverly, MA), and cleaved caspase-3 (Asp175) Rabbit pAb (Cell Signaling, Beverly, MA). The membranes were developed on a Molecular Imager ChemiDoc XRS+ System (Bio-Rad, Hercules, CA) and the intensity of the protein band was analyzed by ImageJ software (1.46b, National Institutes of Health, USA). All Western blot analyses were performed in triplicate.
Acetyl histone h3
Manufactured by Merck Group
Sourced in United States, United Kingdom
Acetyl-histone H3 is a laboratory product that is used to detect and analyze acetylation of histone H3 protein, a key component of chromatin. It serves as a tool for epigenetic research and studies related to histone modifications.
Automatically generated - may contain errors
Lab products found in correlation
Acetyl histone h4, by Merck Group (13 mentions)
β actin, by Merck Group (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Chromatin immunoprecipitation kit, by Merck Group (3 mentions)
Actin a5441, by Merck Group (2 mentions)
Ki 67 ab8191, by Abcam (2 mentions)
Jmjd1a 12835 1 ap, by Proteintech (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Vibra cell, by Sonics (2 mentions)
Strataclean resin, by Agilent Technologies (1 mentions)
Ponceau red stain, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
42 protocols using acetyl histone h3
1
Quantitative Protein Expression Analysis
2
Quantifying Fibrotic Lung Protein Levels
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Fibroblasts were cultured as for RNA analysis. After incubation, cells were lysed into SDS sample buffer for SDS-PAGE and cell conditioned media clarified by centrifugation before concentrating secreted proteins using StrataClean resin at 1:10 (Agilent Technologies, Wokingham, UK) [54 (link)]. For murine fibrosis studies, right lungs were snap frozen and then homogenized in normal saline containing protease inhibitors (Roche, Burgess Hill, UK) before solubilization in SDS sample buffer for SDS-PAGE.
Western blotting of cellular or lung lysates was performed for acetyl histone H3, 1:20000 (Merck Millipore, Watford, UK), and of cell-conditioned medium for secreted pro-LOX and LOX, (1:20, Sigma-Aldrich, Poole, UK) using enhanced chemiluminescence detection (ECL+, GE Healthcare, Buckinghamshire, UK). In the case of acetyl H3, membranes were stripped and reprobed using pan histone H3 antibodies (Merck Millipore, Watford, UK); for LOX quantitation, image analysis of Ponceau Red stain (Sigma-Aldrich, Poole, UK) was used a loading control. Densitometry was used for semi-quantitative analysis of the data.
Western blotting of cellular or lung lysates was performed for acetyl histone H3, 1:20000 (Merck Millipore, Watford, UK), and of cell-conditioned medium for secreted pro-LOX and LOX, (1:20, Sigma-Aldrich, Poole, UK) using enhanced chemiluminescence detection (ECL+, GE Healthcare, Buckinghamshire, UK). In the case of acetyl H3, membranes were stripped and reprobed using pan histone H3 antibodies (Merck Millipore, Watford, UK); for LOX quantitation, image analysis of Ponceau Red stain (Sigma-Aldrich, Poole, UK) was used a loading control. Densitometry was used for semi-quantitative analysis of the data.
3
Protein Extraction and Western Blotting Protocol
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A detailed description of protein extraction and Western blotting procedures can be found in our previous report [5 (link)].
In brief, muscle samples were loaded and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Sanford, ME, USA, #sc-3724), after which membranes were incubated in a blocking buffer (TBS-T: 4% non-fat milk powder; Tris-buffered saline, pH 7.4; and 0.1% Tween 20). The membranes were then incubated with primary and secondary antibodies and washed in TBS-T. The primary antibodies used were GAPDH (1:10,000, Applied Biological Materials Inc., Richmond, BC, Canada, # G041), Lamin B1 (1:500, Abcam, Cambridge, MA, USA, # ab16048), MEF2-D (1:1000, EMD Millipore, Temecula, CA, USA, # AB2263), acetyl-Histone H3 (1:1000, EMD Millipore, Temecula, CA, USA, # 06-599), total Histone H3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, # 9715), HDAC4 (1:500, Cell Signaling, Danvers, MA, USA, #2072), HAT P300 (1:500, Abcam, Cambridge, MA, USA, # ab231010).
Secondary HRP-conjugated antibodies (1:30,000) to rabbit or mouse immunoglobulins were from Santa Cruz Biotechnology, CA, USA. Protein bands were detected and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA, #170-5061) and C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA).
In brief, muscle samples were loaded and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Sanford, ME, USA, #sc-3724), after which membranes were incubated in a blocking buffer (TBS-T: 4% non-fat milk powder; Tris-buffered saline, pH 7.4; and 0.1% Tween 20). The membranes were then incubated with primary and secondary antibodies and washed in TBS-T. The primary antibodies used were GAPDH (1:10,000, Applied Biological Materials Inc., Richmond, BC, Canada, # G041), Lamin B1 (1:500, Abcam, Cambridge, MA, USA, # ab16048), MEF2-D (1:1000, EMD Millipore, Temecula, CA, USA, # AB2263), acetyl-Histone H3 (1:1000, EMD Millipore, Temecula, CA, USA, # 06-599), total Histone H3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, # 9715), HDAC4 (1:500, Cell Signaling, Danvers, MA, USA, #2072), HAT P300 (1:500, Abcam, Cambridge, MA, USA, # ab231010).
Secondary HRP-conjugated antibodies (1:30,000) to rabbit or mouse immunoglobulins were from Santa Cruz Biotechnology, CA, USA. Protein bands were detected and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA, #170-5061) and C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA).
4
Antibody Sources for Protein Analysis
5
Cryosectioning and Immunostaining of Enucleated Eyeballs
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Each enucleated eye ball was fixed with paraformaldehyde and then placed in a slurry of optimal cutting temperature (OCT) compound in cryomold before freezing in dry ice and storage in a − 80 °C freezer until ready for sectioning using the Microm HM550 (Carl Zeiss Ltd). Five-micrometer cryosections of day 2 post-operated eye tissues stained with hematoxylin and eosin (H&E) staining was visualized as described previously [14 (link)]. A total of 3 eyes for each condition were evaluated. Acetyl-histone H3, CD45 and F4/80 antibodies were obtained from Merck Millipore (Darmstadt, Germany), BD Pharmingen (San Diego, CA, USA) and Abcam Plc (Cambridge, UK), respectively. Isolectin B4-Alexa Fluor 568 conjugate was from Molecular Probes Inc. (Eugene, OR, USA). Labeling by the primary antibodies was detected using secondary antibodies conjugated to Alexa Fluor-594 (red fluorescence) or Alexa Fluor-488 (green fluorescence), both obtained from Invitrogen Corp. (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclei were visualized by mounting the cells in DAPI-containing Vectashield mounting medium (Vector Laboratories, CA, USA). Labeled cells were visualized using the Zeiss Imager.Z1 microscope (Carl Zeiss Inc., USA).
6
Antibody Sources for DNA Damage Research
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Antibodies were purchased from the following companies: JMJD1A (12835-1-AP) was from Proteintech (Rosemont, IL). HUWE1 (A300-486A) was from Bethyl Laboratories (Montgomery, TX). RNF8 (sc-271462), BRCA1 (sc-6954), p300 (sc-585), ubiquitin (sc-6085), GFP (sc-8334), HA (sc-7392) and c-Myc (sc-789, sc-40) were from Santa Cruz Biotechnology (Dallas, TX). c-Myc (ab56), PRKDC (ab32566) and Ki67 (ab8191) were from Abcam (Cambridge, UK). NBS1 (3002), p-ATM (4526), ATM (2873), p-Chk2 (2197), Chk2 (2662), XRCC6 (4588), and active caspase-3 (9661) were from Cell Signaling (Danvers, MA). γ-H2AX (05-636), ubiquitin (FK2, ST1200), 53BP1 (MAB3802), Rad51 (ABE257), H3K9me2 (07-441) and acetyl-histone H3 (06-599) were from EMD Millipore (Burlington, MA). Flag (F7425, F3165) and actin (A5441) were from Sigma-Aldrich (St. Louis, MO). Etoposide was from Sigma-Aldrich (St. Louis, MO). Olaparib was from MedChemExpresss (Monmouth Junction, NJ).
7
Western Blot Analysis of Histone Acetylation
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Both the treated and untreated HCF cultures were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) followed by centrifugation at 10,000 g for 10 min. Samples were suspended in a NuPAGE LDS buffer containing a reducing agent (Life Technologies Corporation, Grand Island, NY) and heated at 70 °C for 10 min. Protein samples were resolved by NuPAGE Novex Bis-Tris mini gels (Invitrogen) and transferred onto the polyvinylidene difluoride membranes using wet transfer at 25 V. The transferred proteins were detected by incubating the membrane with primary antibodies: TGIF1, TGIF2 (Santa Cruz Biotechnology, Santa Cruz, CA), acetylhistone H3, acetyl histone H4 (EMD Millipore, Billerica, MA), alpha smooth muscle actin (αSMA Dako, Carpinteria, CA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), followed by alkaline phosphatase conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibody. After washing three times in 0.05% Tween-20 in Tris-buffered saline of pH 8.0 for 5 min each, the blot was developed using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate method. Three separate western blots were performed for each experiment. The digital quantification of western blots was performed using NIH Image J software.
8
Western Blot Analysis of Cell Lysates
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Cells were harvested by centrifugation, washed with PBS and lysed using buffer composed of 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1.0% NP-40, 0.1% SDS, supplemented with protease and phosphatase inhibitors. Protein concentrations were estimated by Bradford assay and equivalent quantities of the lysates were resolved on 4–20% Tris-HCl SDS-PAGE TGX gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes and stained for acetyl-histone H3 (Milipore), acetyl-histone H4 (Milipore), IGF2BP1 (Cell Signaling Technology), IGF2BP3 (IMP-3, Santa Cruz Biotechnology), CD44 (Santa Cruz Biotechnology), CD48 (Abcam), Drosha (Cell Signaling Technology), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology), followed by anti-mouse, or anti-rabbit IgG-HRP (GE Healthcare). Signals were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).
9
ChIP Analysis of Histone Modifications and SIRT1
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ChIP analysis was performed using the simpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Briefly, cells were crosslinked with 1% formaldehyde and nuclear DNA was then digested with micrococcal nuclease. The chromatin fractions were extracted. The resulting samples were subjected to immunoprecipitation using polyclonal antibodies to Acetyl-histone H3 (5μl/assay, millipore), Acetyl-histone H4 (5μl/assay; millipore), or SIRT1 (5μl/assay, Cell Signaling Technology). The immunoprecipitated DNAs were quantified by real-time RT-PCR analysis. PCR was performed using the following primers.
IL-8 promoter region; Forward: 5′-GGTTTGCCCTGAGGGGATG-3′ and Reverse: 5′-ACAGAGCTGCAGAAATCAGGAAGGCT-3′, IL-6 promoter region; Forward: 5′-AATGTGGGATTTTCCCATGA-3′ and Reverse: 5′-GCTCCTGGAGGGGAGATAGA-3′.
IL-8 promoter region; Forward: 5′-GGTTTGCCCTGAGGGGATG-3′ and Reverse: 5′-ACAGAGCTGCAGAAATCAGGAAGGCT-3′, IL-6 promoter region; Forward: 5′-AATGTGGGATTTTCCCATGA-3′ and Reverse: 5′-GCTCCTGGAGGGGAGATAGA-3′.
10
Synthesis and Characterization of Azatyrosine-PBHA
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Azatyrosine-PBHA (AzP, Figure 1 A) was synthesized using a patented method (US Patent Grant US 8242282 B2). The dry powder was collected and stored at 4 °C. In in vitro models, AzP was readily dissolved in DMSO; while in the in vivo models, AzP was dissolved with hydroxypropyl beta-cyclodextrin (HPβCD), a sugar derivative of good solubilizing capacity, in a 1:10 ratio.
Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12K medium, penicillin/streptomycin/glutamate and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY, USA). The horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) Ab and HRP-conjugated anti-rabbit IgG Ab were obtained from Amersham Biosciences (Sunnyrale, CA, USA). Except for acetyl-Histone H3 (Millipore, San Diego, CA, USA), other primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All of the other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12K medium, penicillin/streptomycin/glutamate and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY, USA). The horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) Ab and HRP-conjugated anti-rabbit IgG Ab were obtained from Amersham Biosciences (Sunnyrale, CA, USA). Except for acetyl-Histone H3 (Millipore, San Diego, CA, USA), other primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All of the other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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