Chemicals and drugs of the purest available grade including NAC were provided by Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies anti-P-AKT (reacting with Ser473), anti-AKT, anti-Insulin-receptor (anti-IR), anti-fructokinase (anti-fructokinase), anti-glutathione-peroxidase (anti-GPx), and anti-glutathione reductase (anti-GR) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; catalog number 6040S, 9272,sc-711, sc-50029, sc-133160, and sc-133245, respectively). Anti-COX-2 from CAYMAN Laboratories (Ann Arbor, MI, USA catalog number 160106), anti-iNOS, and anti-eNOS were obtained from Sigma (St. Louis, MO, USA; catalog number N7782). Anti-P-eNOS (Ser 1177) was provided by Cell Signaling Laboratory (Danvers, MA, USA; catalog number N3893); anti-glucokinase antibody (sheep anti-GST-glucokinase fusion protein antibody) was kindly provided by Dr. Mark Magnusson (Vanderbilt University, TN, USA). This antibody, another from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA; glucokinase-N-19: sc:1980), and anti-GAPDH from Millipore (Carlsbad, CA, USA; catalog number 92590) were also provided. Finally, a secondary antibody anti-rabbit IgG Peroxidase (developed in goats) was obtained from Sigma (St. Louis, MO, USA; catalog number A9169).
Anti rabbit igg peroxidase
Manufactured by Merck Group
Sourced in United States, Japan
Anti-rabbit IgG-peroxidase is a laboratory reagent used in immunoassays and other biochemical applications. It consists of anti-rabbit immunoglobulin G (IgG) antibodies conjugated to the enzyme peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg peroxidase, by Merck Group (12 mentions)
Anti gapdh, by Merck Group (2 mentions)
P stat5, by Cell Signaling Technology (2 mentions)
Anti goat igg peroxidase, by Merck Group (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Anti p akt, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Hdac1, by Merck Group (1 mentions)
Histone h3, by Abcam (1 mentions)
Hdac3, by Cell Signaling Technology (1 mentions)
Stat5a, by Santa Cruz Biotechnology (1 mentions)
Stat5b, by Santa Cruz Biotechnology (1 mentions)
Acetylated histone h3 ac h3, by Merck Group (1 mentions)
Acetylated histone h4 ac h4, by Merck Group (1 mentions)
Hdac2, by Thermo Fisher Scientific (1 mentions)
Rna polymerase 2, by Santa Cruz Biotechnology (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
31 protocols using anti rabbit igg peroxidase
1
Detailed Protein Analysis Methodology
2
Antibody Validation for Western Blot and ChIP
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Antibodies used for western blot and ChIP were pSTAT5 (Cell Signaling Technology 9351), STAT5A (Santa Cruz Biotechnology sc-1081), STAT5B (Santa Cruz Biotechnology sc-1656), STAT5A+B (Santa Cruz Biotechnology sc-835), RNA polymerase II (Santa Cruz Biotechnology sc-899 and sc-900), TBP (Santa Cruz Biotechnology sc-273), histone H3 (Abcam ab1791), acetylated histone H3 (Ac-H3; Millipore 06–599), acetylated histone H4 (Ac-H4; Millipore 06–866), α-tubulin (Santa Cruz Biotechnology sc-32293), HDAC1 (Millipore 05–100), HDAC2 (Invitrogen 51–5100), HDAC3 (Cell Signaling Technology 2632), FLAG (M2, SIGMA F-1804), Brd2 (Bethyl A302–583A) and IgG from rabbit serum (SIGMA I-5006; isotype control for ChIP). Secondary antibodies for western blot were anti-Rabbit IgG-Peroxidase (SIGMA A-0545) and anti-Mouse IgG-Peroxidase (SIGMA A-8924).
3
Western Blot Analysis of C. crescentus
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C. crescentus swarmer cells were isolated using the mini-synchrony protocol, and the inducer was added (as specified in the figure legends). OD600 of incubated cultures were normalized to ~0.2, and the cell pellets resuspended in Cracking buffer (40 μL) were boiled at 80°C for 10 min and stored at –20°C for Western blots. Proteins were separated on SDS-PAGE and transferred into the nitrocellulose membrane using iBlot2 (Invitrogen Dry Blotting System). The membrane was blocked with 1 × TBS (10 mM Tris-Cl, pH 8.0, 150 mM NaCl) with 5% nonfat milk and 0.1% Tween 20 (T) for 1 h. The blot was probed with ~1:10,000 diluted primary antibody (anti-Flag for ParA-M2, F7425 Sigma-Aldrich, and anti-DnaA for DnaA) overnight at 4°C. The membrane was washed three times with 1 × TBST and incubated for 1 h with 1:10,000 diluted secondary antibody (Anti-Rabbit IgG peroxidase, Sigma-Aldrich) at room temperature. Excess secondary antibody was removed by washing the membrane with 1 X TBST three times. The membrane was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific), and imaged with ChemiDoc-MP imaging system (Bio-Rad Laboratories).
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4
Fibrosis Signaling Pathway Protocols
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PDE5-I vardenafil was obtained from Sequoia Research Products Ltd. (vardenafil citrate), Sigma Aldrich, or Selleckchem (vardenafil HCl). Nintedanib was obtained from Cayman Chemical. TGF-β1 was obtained from R&D Systems. The antibodies used in this study were as follows: fibronectin (Sigma Aldrich, F3648, St. Louis, MO, USA), CTGF (Santa Cruz Biotechnology, sc-14939, Dallas, TX, USA), αSMA (Sigma, A2547), p-SMAD3 (Cell Signaling 95205, Danvers, MA, USA), total SMAD3 (Abcam AB 28379), and GAPDH (Millipore MAB374). The secondary antibodies used for fibronectin ELISA included anti-rabbit IgG peroxidase (Sigma Aldrich, A0545) and anti-rabbit IgG-HRP (SantaCruz Biotechnology sc-2004). Additional antibodies for TGF-β1 signaling included the following: anti-mouse serpin E1/PAI-1 antibody (AF3828; R&D systems), anti-CTGF (sc-14939; SCBT), anti-αSMA (A2547; Sigma), anti-fibronectin (F3648, Sigma), anti-GAPDH (AB2302; Millipore, Burlington, MA, USA), anti-phospho-SMAD3 and anti-phospho-SMAD2 antibodies generated in our laboratory, anti-SMAD2 (ab63576; Abcam, Cambridge, UK), anti-SMAD3 (ab28379; Abcam), anti-phospho-AKT (Ser473; 9271, Cell Signaling), anti-phospho-AKT (T308; 4056, Cell Signaling), anti-phospho S6K (Thr389, 9234, Cell Signaling), anti-Akt (9272, Cell Signaling), and anti-S6K (9202, Cell Signaling).
5
Western Blot Protein Analysis Protocol
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Total proteins were extracted in RIPA buffer containing 1 mM sodium orthovanadate and Complete Protease Inhibitor Cocktail (Roche Applied Science) and quantified using the Pierce™ BCA protein assay kit (Thermo Fisher). Equal amounts of proteins were loaded onto SDS–polyacrylamide gels and transferred to PVDF membrane. The membranes were then blocked with 5% non‐fat milk in TBST 0.1% and immunoblotted overnight at 4°C with the following primary antibodies (1:1,000): TCHP (Santa Cruz Biotechnology, SC‐515025), β‐actin (Sigma, A5441), p62 (GeneTex, GTX100685), LC3B (CST, #2775), p16 (BD Biosciences, 551154), NF‐κB (Millipore, MAB3026), (S536) NF‐κB (CST, #3033), PCM1 (CST, #5213) and GABARAP (CST, #13733), NDP52 (CST, #60732) and V5 (Thermo Fisher, R960‐25). Secondary antibodies (1:5,000): anti‐Mouse IgG–Peroxidase (Sigma, A5906) and anti‐Rabbit IgG–Peroxidase (Sigma, A0545), were incubated for 1 h at RT. Pixel intensity/quantification was performed using ImageJ.
6
Protein Extraction and Western Blot Analysis
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Total proteins were prepared by resuspending 1 × 106 cells in extraction buffer (50 mM Tris-HCl pH 7.6; 0.15 M NaCl; 5 mM EDTA; 16 Protease Inhibitors; 1% Triton X-100). One 40 s pulse of sonication (UP100H manual sonicator, Hielscher) at 40% amplitude was performed to allow dissociation of protein from chromatin and solubilization. Extracts were analyzed by SDS-PAGE using an 8% gel (37.5:1 Acryl/Bis Acrylamide). The following primary antibodies were used: Beta-Actin (Santa-Cruz sc1616, rabbit 1:4000), H3 total (Abcam ab1791, rabbit 1:6000), Lamin A/C (Santa Cruz sc-6215, goat 1:4000), Lamin B (Santa Cruz sc6216, goat 1:2000), progerin (13A4 mouse, Abcam 66587, mouse 1:1000), Ezh2 (AC22 Cell Signaling 3147S, mouse 1:1000), Bmi1 (D42B3 Cell signaling, rabbit 1:1000), H3K9me3 (Abcam ab8898, rabbit 1:1000), H3K27me3 (Millipore 07-449 rabbit 1:1000). HRP-conjugated secondary antibodies were revealed with the ECL chemiluminescence kit (ThermoFisher Scientific). The following secondary antibodies were used: Anti-Mouse IgG-Peroxidase (Sigma, A9044), Anti-Rabbit IgG-Peroxidase (Sigma, A9169), Anti-Goat IgG-Peroxidase (Sigma, A5420).
7
Phosphoinositide Binding Assay with PIP-Strips
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PIP-strips supplied by Echelon Biosciences, Inc. (Salt Lake City, Utah, USA) were used to perform phosphoinositide binding according to the supplied protocol. Briefly, GST and GST-fusion proteins were eluted from the glutathione agarose beads with elution buffer (20 mM reduced glutathione, 50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.2% Tween-20, and 100 mM DTT).
The membranes were blocked with 0.1% ovalbumin (Sigma # A-5253) in TBS for one hour at room temperature. After discarding the blocking solution membranes were incubated with 1 mg/ml GST-fusion proteins in TBS-T (50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.2% Tween-20) at room temperature for one hour. The protein solution was then discarded and the membranes were washed with TBS-T three times 10 minutes each. Bound protein was detected by western blot analysis with GST polyclonal antibodies as primary and anti-rabbit IgG-peroxidase (Sigma # A-6154) as secondary antibody followed by enhanced chemiluminescence.
The membranes were blocked with 0.1% ovalbumin (Sigma # A-5253) in TBS for one hour at room temperature. After discarding the blocking solution membranes were incubated with 1 mg/ml GST-fusion proteins in TBS-T (50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.2% Tween-20) at room temperature for one hour. The protein solution was then discarded and the membranes were washed with TBS-T three times 10 minutes each. Bound protein was detected by western blot analysis with GST polyclonal antibodies as primary and anti-rabbit IgG-peroxidase (Sigma # A-6154) as secondary antibody followed by enhanced chemiluminescence.
8
Chromatin-Enriched Protein Extraction Protocol
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Embryos were washed with PBS and lysed on ice for 10 min in a lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM MgCl2, 10% [v/v] glycerol, 0.2% [v/v] NP-40, and 1 mM NaF). Chromatin-enriched fractions were collected by low-speed centrifugation at 1500g for 5 min, washed with the lysis buffer, and further lysed with 1× SDS buffer. Proteins were separated using polyacrylamide gels (5.5% for SMC3 and 12.5% for histone H3) and transferred onto polyvinylidene difluoride membrane (MilliporeSigma). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies (SMC3: abcam ab9263, histone H3: abcam ab1791) overnight at 4°C. After several washes, membranes were incubated with secondary antibody (anti-rabbit IgG Peroxidase, Sigma-Aldrich A0545) for 1 h at room temperature. Protein bands were visualized using the ECL Select Western Blotting Detection Reagent (GE Healthcare) and detected by ImageQuant (GE Healthcare).
9
Metformin Cytotoxicity and Apoptosis Assay
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Metformin (1,1-dimethyl biguanide hydrochloride) was purchased from Sigma (#D150959, Sigma-Aldrich, St. Louis, MO, USA) and dissolved in a serum-free medium to a stock solution of 1 M. Other chemicals and reagents were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and Merck (Merck KGaA, Darmstadt, Germany).
Antibodies were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and CST (Cell Signaling Technology, Beverly, MA, USA). The antibodies used for Western blots were anti-rabbit IgG-peroxidase (#A0545, Sigma), rabbit anti-GAPDH antibody (#G9545, Sigma), rabbit anti-β-actin antibody (#A2066, Sigma), anti-PARP antibody (#9542, CST), anti-caspase-3 antibody (#9662, CST) and anti-cleaved caspase-3 antibody (#9664, CST).
Antibodies were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and CST (Cell Signaling Technology, Beverly, MA, USA). The antibodies used for Western blots were anti-rabbit IgG-peroxidase (#A0545, Sigma), rabbit anti-GAPDH antibody (#G9545, Sigma), rabbit anti-β-actin antibody (#A2066, Sigma), anti-PARP antibody (#9542, CST), anti-caspase-3 antibody (#9662, CST) and anti-cleaved caspase-3 antibody (#9664, CST).
10
Cyclin D1 and DEPTOR Protein Expression Analysis
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For Western Blot analysis, cells were washed with PBS and scraped after addition of denaturing buffer (160 mM Tris-HCl pH 6.8, 4% SDS, 10% b-mercaptoethanol, 24% glycerol and 0.02% bromophenol blue). Protein extracts were subjected to SDS-PAGE, transferred to nitrocellulose membrane and probed with rabbit anti-cyclin D1 (produced in rabbit, Neomarkers, cat#RB-4091-P1, 1:8000) and anti-DEPTOR/DEPDC6 (D9F5) (produced in rabbit, Cell Signaling, cat#11816 S, 1:1000) antibodies. After incubation with secondary antibody anti-rabbit IgG-peroxidase (produced in goat, Sigma, cat#A6154, 1:2500), membranes were analyzed with Novex® ECL HRP Chemiluminescent kit. The signals obtained were quantified with ImageJ software.
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