Proteins in the nuclear extracts (60 μg protein) were separated by SDS–PAGE gels (456–1043, Bio-Rad, Hercules, CA), transferred onto PVDF membranes (162–0174, Bio-Rad), and incubated with anti-histone antibodies: acetylated H3K9 (1/2000: ab4441, abcam, Cambridge, MA), acetylated H3K14 (1/5000: 06–911, EMD Millipore, Billerica, MA), acetylated H4K5 (1/2000: 07–327, EMD Millipore). Bound antibody was detected by horseradish peroxidase-conjugated anti-rabbit secondary antibody (1/20000: 1705046, Bio-Rad) and developed using Clarity™ Western ECL Substrate (1705060, Bio-Rad). Signals were detected and captured by ImageQuant™ LAS 4000 mini (GE healthcare, Pittsburgh, PA), and band intensities were quantified with ImageJ software. H3K9 acetylation intensity in individual lanes was reported relative to the normalized Mock treatment, and calculated using this formula: Relative H3K9ace intensity for each timepoint = (H3K9ace / PCNA) / averaged (Mock H3K9ace / Mock PCNA). Statistical significance was determined by unpaired t test (two-tailed) against mock at each time point.
Horseradish peroxidase conjugated anti rabbit secondary antibody
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit secondary antibody is a reagent used in various immunoassay techniques. It consists of a secondary antibody directed against rabbit primary antibodies, conjugated to the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric reaction, allowing for the detection and quantification of target analytes bound by the primary antibody.
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Lab products found in correlation
Mini protean, by Bio-Rad (3 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Acetylated h3k9, by Abcam (2 mentions)
Acetylated h3k14, by Merck Group (2 mentions)
Acetylated h4k5, by Merck Group (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Las 4000, by Cytiva (2 mentions)
Anti actin, by Merck Group (1 mentions)
Anti h3, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Agrisera (1 mentions)
Anti hif 1α, by Merck Group (1 mentions)
Anti vegf a, by Santa Cruz Biotechnology (1 mentions)
Everyblot blocking buffer, by Bio-Rad (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
10 protocols using horseradish peroxidase conjugated anti rabbit secondary antibody
1
Quantifying Histone Acetylation Dynamics
2
Immunoblotting protocol with antibody detection
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For immunoblotting analysis, samples were separated by SDS-PAGE, performed as described by Laemmli (1970) (link) in a Mini Protean apparatus (Bio-Rad, Hercules, CA), and transferred onto a PVDF membrane with a Bio-Rad semi-dry apparatus following the manufacturer’s instructions. Anti-STRP polyclonal antibody 1:10,000 dilution, anti-H3 (Sigma-Aldrich Corporation) 1:2,000 dilution, anti-H+-ATPase (Camoni et al., 2006 (link)) 1:2,000 dilution and anti-actin (Sigma-Aldrich Corporation) 1:1,000 were used. Immunoblot detection was performed by incubating the membrane with horseradish peroxidase conjugated anti-rabbit secondary antibody (1:5,000) or anti-mouse secondary antibody (1:5,000) from Bio-Rad.
3
SDS-PAGE and Western Blot Analysis
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Proteins were separated on SDS–Page in a Mini Protean apparatus (Bio-Rad, Hercules, CA, USA) [54 (link)] and then electroblotted on PVDF membrane with the Trans–Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Anti-STRP [7 (link)] 1:10,000, anti-actin (Agrisera, Vännäs, Sweden) 1:5000, and anti-GFP (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, DE) 1:1000 dilutions were used. Protein detections were performed by incubating the membrane with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:5000) or anti-mouse secondary antibody (1:5000) from Bio-Rad.
4
Western Blot Analysis of Histone Acetylation
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Proteins in the nuclear extracts (60 μg protein) were separated by SDS-PAGE gels (456-1043, Bio-Rad, Hercules, CA), transferred onto PVDF membranes (162-0174, Bio-Rad), and incubated with anti-histone antibodies: acetylated H3K9 (1/2000: ab4441, abcam, Cambridge, MA), acetylated H3K14 (1/5000: 06-911, EMD Millipore, Billerica, MA), and acetylated H4K5 (1/2000: 07-327, EMD Millipore). Bound antibody was detected by horseradish peroxidase-conjugated anti-rabbit secondary antibody (1/20,000: 1705046, Bio-Rad) and developed using Clarity Western ECL Substrate (1705060, Bio-Rad). Signals were detected and captured using ImageQuant LAS 4000 mini (GE Healthcare, Pittsburgh, PA), and band intensities were quantified with ImageJ software. H3K9 acetylation intensity in individual lanes was reported relative to the normalized Mock treatment and calculated using this formula: relative H3K9ace intensity for each timepoint = (H3K9ace/PCNA)/averaged (Mock H3K9ace/Mock PCNA). Statistical significance was determined by unpaired t-test (two-tailed) against mock at each time point.
5
SDS-PAGE and Immunoblotting Analysis
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SDSePAGE was performed as described by La€ emmli (1970) , in a Mini Protean apparatus from Bio-Rad (Hercules, CA). For immunoblotting analysis, proteins were separated by SDSePAGE, then electroblotted onto a PVDF membrane with 39 mM glycine, 48 mM Tris, 0.1% SDS and 10% methanol. After blocking for 1 h in TTBS (20 mM TriseHCl, pH 7.5, 100 mM NaCl, 0.05% Tween-20) with 5% no-fat dried milk at room temperature, the membrane was incubated with anti-14-3-3 antibodies (1:1000) or anti-H þ -ATPase antibodies (1:1000) (Marra et al., 2000) . Following three washes with TTBS, the membrane was incubated with horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:5000) from Bio-Rad (Hercules, CA).
6
Western Blot Analysis of Retinal Proteins
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For Western blot analysis, retinas were removed 8 weeks after the first injections from each separate group. Samples (n = 16) were processed for Western blot analysis as described earlier [31 (link)]. Protein concentrations were determined using Bradford reagents. Membranes were blocked in EveryBlot Blocking Buffer (BioRad; Hercules, CA, USA) for 5 min at room temperature and were probed at room temperature with anti-HIF1-α (1:2000; Sigma-Aldrich, Budapest, Hungary) and anti-VEGF-A (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Non-phosphorylated anti-GAPDH (1:20000; Cell Signaling Technology; Danvers, MA, USA) was used as internal control. Membranes were washed in Tris-buffered saline (TBS; pH = 7.5) containing 0.2% Tween. Anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3000; BioRad; Hercules; CA, USA) was diluted in EveryBlot Blocking Buffer (BioRad; Hercules; CA, USA) and the membranes were incubated for 1 h at room temperature. The antibody–antigen complexes were visualized by means of enhanced chemiluminescence. For quantification of blots, band intensities were quantified by the NIH ImageJ program (National Institutes of Health, Bethesda, MD, USA).
7
Western Blot Analysis of Eca-109 Cells
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Total protein was extracted on ice from Eca-109 cells with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (catalog no., P2813; Sigma-Aldrich, St. Louis, MO, USA). Membranes were blocked with 2% fat-free milk in phosphate-buffered saline (PBS) at room temperature for 1 h and then probed with primary antibodies (dilution, 1:200) overnight at 4°C in PBS containing 0.1% Tween 20 (PBST) and 1% fat-free milk. Following the overnight incubation, membranes were washed four times in PBST and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:200; catalog no., 1662408; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Signals were developed using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Densitometric analysis was performed to quantify the results using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
8
Isolation and Analysis of PDYN from Nuclei
9
Protein Extraction and Western Blot Analysis
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Gast were frozen in liquid nitrogen and proteins were extracted in RIPA lysis buffer [PBS, 0.1% SDS (Promega), 0.5% Sodium Deoxycholate (Sigma-Aldrich),1% Nonidet NP40 (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich), 1 mM Na3VO4 (Sigma-Aldrich), 20 mM NaF (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), cocktails of Protease inhibitors (Sigma-Aldrich)]. Proteins were denatured and loaded in a 10% SDS-PAGE gels (10 μg) and further transferred onto nitrocellulose PVDF membranes. Membranes were incubated with antibodies overnight (Table S1 ) then washed in TBS-Tween 3‰ and further incubated with anti-Rabbit Horseradish Peroxidase conjugated secondary antibody (#172-1019, Bio-Rad). Signals were revealed with Immuno detection kit ECL Luminata Classico (Millipore) and the imager Molecular Image® ChemiDoc™XRS + (Bio-Rad). Proteins quantification was performed by using ImageLab 3.0 (Bio-Rad).
10
Liver Protein Extraction and Western Blot
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Total proteins were isolated from ~50 mg of snap-frozen liver tissue by homogenizing in 600 μl of cold radioimmunoprecipitation assay buffer containing 10 mM tris-Cl (pH 8), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, 140 mM NaCl, 0.1% SDS, and phosphatase and protease inhibitors (one tablet per 10 ml of buffer; catalog nos. 4906837001 and 4693159001). Samples were sonicated in a water bath to shear DNA and clarified by centrifugation. Protein concentration was measured using the DC (detergent compatible) Protein Assay Kit (Bio-Rad, 5000112). Proteins (~20 μg) were resolved on a 4 to 20% TGX (Tris-Glycine eXtended) gel (Bio-Rad) and transferred semidry using “Mixed MW” program on the Bio-Rad Trans Blot Turbo System onto a nitrocellulose membrane with 0.45-μm pore size (Bio-Rad). Membranes were blocked for 1 hour at room temperature using tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 (TBST). Membranes were then incubated with primary antibody against albumin (1:1000; Cell Signaling Technology, no. 4929) overnight at 4°C. The membranes were then washed thrice with TBST, followed by incubation with an anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:5000; Bio-Rad, no. 1706515) for 1 hour. Membranes were then visualized on the ChemiDoc MP using the Clarity Western ECL Kit (Bio-Rad).
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