The rabbit anti-APAP-AD antibody was a kind gift from Dr. Lance Pohl (National Heart, Lung and Blood Institute).31 (link) The other antibodies were: CYP2E1 (Abcam, #ab28146),p62 (Abnova, #H00008878-M01), phospho-eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1) (Cell Signaling, #9451), total 4EBP1 (Cell Signaling, #9452), p-JNK (Cell Signaling, #4668), total JNK (BD Pharmingen, #554285), β-actin (Sigma, #a5541), and glyceralde-hyde phosphate dehydrogenase (GAPDH) (Cell Signaling, #2118). The microtubule-associated protein 1 light chain 3 (LC3) antibody was developed as described previously.36 The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat-anti-rabbit (Jackson ImmunoResearch, #111–035-045), and HRP-conjugated goat-anti-mouse (Jackson ImmunoResearch, #115-035-062).
Horseradish peroxidase hrp conjugated goat anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit is a laboratory reagent used for detecting and quantifying target proteins in various immunoassays. It consists of an antibody raised in goats against rabbit immunoglobulins, which is covalently coupled to the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse, by Jackson ImmunoResearch (4 mentions)
Cyp2e1, by Abcam (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Total jnk, by BD (2 mentions)
Total 4e bp1, by Cell Signaling Technology (2 mentions)
P 4ebp1, by Cell Signaling Technology (2 mentions)
Pi4kiiiβ, by BD (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Comet assay kit, by Bio-Techne (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Lmagarose, by Bio-Techne (1 mentions)
2 well cometslides, by Bio-Techne (1 mentions)
Sybr green, by Bio-Techne (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Glutathione agarose, by Merck Group (1 mentions)
Hilyte plus 647 conjugated goat anti mouse igg, by AnaSpec (1 mentions)
Alexa fluor 555 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Myc epitope tag, by Merck Group (1 mentions)
15 protocols using horseradish peroxidase hrp conjugated goat anti rabbit
1
Antibody Characterization for APAP Toxicity
2
TRPM2 Pathway Activation Analysis
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Opti-MEM reduced serum medium and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, USA). Protease inhibitor cocktail tablets (Complete Mini EDTA-free) were purchased from Roche (Mannheim, Germany). Primary antibodies utilized were poly-clonal rabbit anti-human TRPM2 antibody (cat. #A300-414A; Bethyl Laboratories, Montgomery, TX, USA), polyclonal rabbit anti-human β-actin (cat. #600-401-886), polyclonal rabbit anti-human apoptosis-inducing factor (AIF) (cat. #200-401-985) (both from Rockland Immunochemicals, Limerick, PA, USA) and monoclonal mouse anti-human poly(ADP-ribose) glycohydrolase (PARG) clone D8B10 (cat. #MABS61; Millipore). Two secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse were purchased from Jackson ImmunoResearch (West Grove, PA, USA). The CometAssay kit, which includes alkaline lysis solution, LMAgarose, 2-well CometSlides and SYBR-Green was purchased from Trevigen (Gaithersburg, MD, USA).
3
Monitoring T7-tagged Protein Interactions
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Monoclonal Anti-T7 IgG (Cat #69522-3) was from Novagen. Hilyte Plus 647-conjugated goat anti-mouse IgG (Cat #AS-61057-05-H647) was from Anaspec (Fremont, CA). Horseradish-peroxidase (HRP)–conjugated goat anti–rabbit (Cat # 111-035-003) or anti–mouse (Cat # 115-035-166) IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Polyclonal Anti-Rab12 (Cat # 18843-1-AP) was from Proteintech (Chicago, IL). Hybridoma IgE was from Myeloma IgE producing cells (clone Hi 26.86), a kind gift from Dr. Ullrich Blank (Inserm, Paris, France). Glutathione-Agarose (Cat # G4510) and guanosine 5′-[γ-thio] thriphosphate (Cat # G8634) were from Sigma-Aldrich (St. Louis, MO). Dinitrophenyl (DNP)-conjugated human serum albumin (Cat #A6661) was from Sigma-Aldrich Chemicals Co (St. Louis, MO). Peptides 1 and 2 were synthesized by the Blavatnik Center for Drug Discovery, at Tel Aviv University.
4
Antibody Characterization for p14 and 14
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Vero and QM5 cells were maintained at 37˚C and 5% CO2 in medium 199 with Earle's salts supplemented with 5 or 10% fetal bovine serum (FBS), respectively. The rabbit polyclonal anti-p14 and anti-14 ectodomain antisera were previously described (Corcoran and Duncan, 2004 (link); Top et al., 2005 (link)). Antibodies against actin (Sigma-Aldrich, St. Louis, MO), myc epitope tag (Sigma-Aldrich), PI4KIIIβ (BD Biosciences, Franklin Lakes, NJ), TGN46 (Serotec, Oxford, UK), protein disulfide isomerase (PDI; Enzo Life Sciences, Farmingdale, NY), horseradish peroxidase (HRP)–conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488–conjugated goat anti-mouse or donkey anti-sheep, Alexa Fluor 555–conjugated donkey anti-rabbit, and Alexa Fluor 647–conjugated goat anti-rabbit secondary antibodies (Life Technologies, Carlsbad, CA) were obtained from the indicated suppliers.
5
Western Blot Analysis of Protein Expression
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Cells were collected by centrifugation and the cell pellet was resuspended in Laemmli sample buffer containing 10% β-mercaptoethanol. Whole cell lysates were boiled for 10 minutes, loaded onto 10% polyacrylamide gel, and proteins were fractioned by SDS-PAGE. Proteins were electrophoretically transferred from gel slabs to nitrocellulose membranes for 90 minutes at 100 V. Membranes were incubated overnight at 4 °C with either rabbit polyclonal IgG that recognizes GFP (1:3000 dilution) or rabbit polyclonal IgG that recognizes GAPDH (1:5000 dilution, Cell Signalling Technology, Danvers, MA). Blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000 dilution, Jackson Immunoresearch Laboratories, West Grove, PA). To visualize proteins, membranes were incubated with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Pittsburgh, PA) and imaged using a Biorad Chemidoc (Biorad, Hercules, CA).
6
Western Blot Analysis of Signaling Pathways
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Primary antibodies: mouse anti-PARP-1, rabbit anti-phospho-Ser473 Akt, rabbit anti-phospho-Thr308 Akt, rabbit anti-Akt, rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-MEK1/2, mouse anti-MEK1/2, mouse anti-p21waf1, rabbit anti-p27, were from Cell Signaling Technologies (Boston, MA, USA). Mouse anti-human GAPDH and rabbit anti-cyclin A were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies: horseradish peroxidase (HRP)—conjugated goat anti-rabbit and goat anti-mouse IgG (H + L) antibodies were from Jackson (Baltimore Pike West Grove, PA, USA). EZ-ECL enhanced chemiluminescence detection kit, RPMI1640 medium, L-glutamine, fetal bovine serum, trypsin, antibiotics and phosphate buffered saline (PBS) were from Biological Industries (Beit-Ha-Emek, Israel). Propidium iodide, phosphatase inhibitor cocktails 2 and 3, sulphorodamine B (SRB), trichloroacetic acid and acetic acid were from Sigma Aldrich (St. Louis, MO, USA). ZSTK474, Selumetinib and AEW-541 were from Selleckchem (Houston, TX, USA). Complete mini protease inhibitor cocktail, RNAse A and Triton X-100 were from Roche Diagnostics Gmbl (Mannheim, Germany).
7
Western Blot Analysis of Liver Proteins
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Proteins were obtained from liver tissues and liver cells using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), while protein concentration was assayed using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were separated using sodium salt-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated overnight with primary antibodies at 4°C (anti-TNF-α [1:1000; Bioss]; anti-OPN [1:1000; Abcam]; anti-CYP7A1 antibodies [1:1000; Invitrogen]; anti-β-actin [1:2000; Abcam]). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:10,000; Jackson, PA, USA) was used as the secondary antibody. Finally, membranes were visualized using enhanced chemiluminescence (Beyotime) and analyzed on Image J 1.8.0 (National Institutes of Health, Bethesda, MD, USA).
8
Investigating Unfolded Protein Response Pathways
9
Autophagy Modulation by α-Asarone
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Dulbecco's Modified Eagle Medium (DMEM) chemicals, monodansylcadaverine (MDC) and 7β-hydroxycholesterol were obtained from Sigma Aldrich Chemical (St. Louis, MO) as were all other reagents, unless specifically stated elsewhere. Fetal bovine serum (FBS) and penicillin-streptomycin were provided by Lonza (Basel, Switzerland). α-Asarone was purchased from Cayman chemical company (Ann Arbor, MI). Antibodies of eIF2α, phospho-eIF2α, Vps34, Atg5, Atg12-Atg5, Atg16L1, SQSTM1, NBR1, Atg7, Atg3, CHOP were obtained from Cell Signaling Technology (Danvers, MA). GADD34, beclin-1 antibodies were purchased from Abcam (Cambridge, UK). p150 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). LC3 antibody was purchased from MBL international corporation (Woburn, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and rabbit anti-mouse IgG were supplied from Jackson ImmunoResearch Laboratory (West Grove, PA).
10
Immunostaining of HeLa and HEK cells
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HeLa cells were maintained in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Life Technologies). HEK cells were maintained in DMEM supplemented with 10% FBS and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Life Technologies). Rabbit polyclonal anti-p14 and anti-p14 ectodomain (anti-p14ecto) antibodies were previously described (Corcoran and Duncan, 2004 (link); Top et al., 2005 (link)). Antibodies against actin (Sigma-Aldrich, St. Louis, MO), Rab11A (BD Biosciences, Franklin Lakes, NJ), AP-1γ (Sigma-Aldrich), AP-3δ (Developmental Studies Hybridoma Bank, Iowa City, IA), AP-4ε (Abcam, Cambridge, MA), PI4KIIIβ (BD Biosciences), TGN46 (AbD Serotech, Oxford, UK), horseradish peroxidase (HRP)–conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA), HRP-conjugated goat anti-mouse (Santa Cruz Biotechnology, Dallas, TX), Alexa 488–conjugated goat anti-mouse and donkey anti-sheep, Alexa 555–conjugated donkey anti-rabbit, and Alexa-647–conjugated goat anti-rabbit and chicken anti-mouse (Life Technologies) were obtained from the indicated suppliers.
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