NT2 cells (the NTera2 human embryonal carcinoma cell line, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen/Life Technologies, Grand Island, NY, USA), supplemented to 10% v/v with FBS. Cells were exposed to 30-ng/ml IL-1β, or to IL-1β plus 10-nM PNR502, for 24 h. The cells were harvested and their proteins extracted in lysis buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 1% w/v Nonidet P40, 0.1% SDS, 0.5% sodium deoxycholate) and quantified with Bradford reagent (Bio-Rad). Protein aliquots (100 μg) were electrophoresed 2 h at 100 V on a 4%–20% gradient bis-tris acrylamide gel (BioRad Life Science, Hercules CA, USA), and transferred to nitrocellulose membranes. Blots, after pre-incubation with BSA blocker (Pierce), were probed with rabbit antibody to active human p38/MAPK (Cell Signaling, 1:100 dilution) or goat anti-human ApoE (Calbiochem) overnight at 4°C. After washes, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibody (AbCam, 1:10,000 dilution) or rabbit anti-goat IgG (Rockland Immunochemicals, Gilbertsville, PA, USA) for 1-h at room temperature. After washes, the membrane was developed with ECL chemiluminescence detection kit (Pierce). Data were digitized and analyzed using ImageJ software (NIH).
Goat anti rabbit igg
Manufactured by Rockland Immunochemicals
Sourced in United States
Goat anti-rabbit IgG is an immunoglobulin G (IgG) antibody produced in goats and directed against rabbit IgG. It is commonly used in immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, as a secondary antibody to detect and visualize the presence of rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Bradford reagent, by Bio-Rad (2 mentions)
Gradient bis tris acrylamide gel, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sheep anti mouse igg, by Rockland Immunochemicals (1 mentions)
Lysis buffer, by Applygen (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti type 1 collagen, by Merck Group (1 mentions)
Anti e cadherin, by Merck Group (1 mentions)
Anti desmin, by Merck Group (1 mentions)
Anti dnmt1, by Merck Group (1 mentions)
Anti smad7, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Phospho eif2α, by Cell Signaling Technology (1 mentions)
10 protocols using goat anti rabbit igg
1
Quantifying p38/MAPK and ApoE Proteins in IL-1β-Treated NT2 Cells
2
Western Blot Analysis of Glioblastoma Cells
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Human glioblastoma cells (T98G) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen/Life Technologies, Grand Island, NY, USA), supplemented to 10% FBS, v/v. Cells were harvested, and their proteins extracted in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% w/v Nonidet P40, 0.1% SDS, 0.5% sodium deoxycholate) and quantified with Bradford reagent (Bio-Rad). Protein aliquots (50 μg) were electrophoresed for 2 h at 100 V on a 4–20% gradient bis-tris acrylamide gel (BioRad Life Science, Hercules, CA, USA) and transferred to nitrocellulose membranes. After pre-incubation with BSA blocker (Pierce/ThermoFisher, Waltham MA, USA), membranes were probed with rabbit antibody to pGFAP or ROCK-1 (Cell Signaling, Danvers MA, USA; 1:100 dilution) overnight at 4 °C. After washes, membranes were incubated for 1 h at room temperature with a secondary antibody, either HRP-conjugated goat anti-rabbit IgG (AbCam, Boston MA, USA; 1:10,000 dilution) or rabbit anti-goat IgG (Rockland Immunochemicals, Gilbertsville, PA, USA), and developed using an ECL chemiluminescence detection kit (Pierce). Data were digitized and analyzed using ImageJ software (NIH).
3
Western Blot Analysis of Muscle and Spinal Cord
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Frozen muscle tissue or spinal cord (L6-S1) tissue was homogenized in ice-cold lysis buffer (Applygen Technologies Inc., Beijing, China) with protease inhibitor cocktail (Roche, Switzerland). Equal amounts (50 μg) of extract were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, United States) at 4°C. Non-specific binding sites were blocked with 10% non-fat milk in TBS (20 mM Tris-Cl, pH 7.5, containing 0.15 M NaCl and 2.7 mM KCl) for 1 h at room temperature. The membrane was incubated with the primary antibodies listed in Table 1 overnight at 4°C. After washes with TBST, the membrane was incubated with the appropriate secondary antibodies goat anti-rabbit IgG (1:10,000, 611-132-122, Rockland, United States) or sheep anti-mouse IgG (1:10,000, 610-632-002, Rockland, United States) for 2 h at room temperature. The membrane was washed in TBST and visualized with an Odyssey Infrared Imager (LI-COR, NE, United States). The integrated intensity of the bands was analyzed with Odyssey software (version 1.2).
4
Immunofluorescent Receptor Labeling
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For each receptor analyzed, two different primary antibodies were tested (Table 1 ). All antibodies used bind extracellular epitopes of their target receptors. Secondary antibodies conjugated to ATTO 647N, goat anti-mouse IgG (# 610-156-121) and goat anti-rabbit IgG (# 611-156-122) were from Rockland Immunochemicals Inc. (Gilbertsville, PA, USA).
Cells on coverslips were briefly washed with complete DMEM medium and incubated with blocking solution (complete DMEM supplemented with 2% bovine serum albumin) for 10 minutes at 37°C and 5% CO2. Coverslips were carefully removed and incubated for 1 hour with primary antibody (1:100) in complete DMEM at 37°C and 5% CO2. After incubation, coverslips were washed with large volume of ice-cold DPBS and cells were then fixed with ice-cold 4% PFA on ice for 20 minutes followed by 30 minutes at room temperature. Remaining aldehydes were quenched for 15 min with 0.1 M glycine in DPBS. Mild permeabilization was performed for 10 min using DPBS supplemented with 2% BSA and 0.1% Triton X-100. Secondary antibodies (1:500) were incubated for 60 minutes at room temperature in a humid chamber protected from light. After incubation, cells were thoroughly washed with DPBS and mounted in Mowiol. Slides were stored at 4°C in the dark until imaging.
Cells on coverslips were briefly washed with complete DMEM medium and incubated with blocking solution (complete DMEM supplemented with 2% bovine serum albumin) for 10 minutes at 37°C and 5% CO2. Coverslips were carefully removed and incubated for 1 hour with primary antibody (1:100) in complete DMEM at 37°C and 5% CO2. After incubation, coverslips were washed with large volume of ice-cold DPBS and cells were then fixed with ice-cold 4% PFA on ice for 20 minutes followed by 30 minutes at room temperature. Remaining aldehydes were quenched for 15 min with 0.1 M glycine in DPBS. Mild permeabilization was performed for 10 min using DPBS supplemented with 2% BSA and 0.1% Triton X-100. Secondary antibodies (1:500) were incubated for 60 minutes at room temperature in a humid chamber protected from light. After incubation, cells were thoroughly washed with DPBS and mounted in Mowiol. Slides were stored at 4°C in the dark until imaging.
5
Protein Expression Analysis Protocol
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Cells were dissociated in cracking 50 mM Tris-HCl (pH 7.4) buffer containing 2-mercaptoethanol (100 mM), SDS (2% w/v), and glycerol (10%). After separation using SDS-PAGE, proteins were electrotransferred onto PVDF membranes (Millipore, USA). The membranes were sealed in milk and incubated with primary antibodies, including anti-type I collagen, anti-E-cadherin, anti-Desmin, anti-Smad7, anti-DNMT1, and anti-β-actin (Sigma, St Louis, MO, USA) antibodies, at 4°C overnight. Subsequently, they were incubated with the secondary antibody, goat anti-rabbit IgG (1:2000, Rockland), for almost 1 h at 37°C. β-actin protein levels were used as the internal control.
6
Investigating Cellular Stress Pathways
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β-ACTIN, CLEAVED-CASPASE3, PERK, phospho-eIF2α, ATF4 rabbit antibodies were purchased from Cell Signaling Technology, (USA). Phospho-PERK antibody was purchased from Abcam (USA), Light Chain 3B-LC3B rabbit antibody was purchased from Novus (USA). B cell lymphoma leukemia2-BCL2 rabbit antibody was purchased from Santa Cruz Biotechnology (USA). The secondary antibody, goat anti-rabbit IgG, was purchased from Rockland (USA). GANT61 were purchased from Sigma Chemical Co (USA), GSK2606414 were purchased from Sellckchem (USA).
7
Immunofluorescent Imaging of CaSR in Cardiomyocytes
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After treatments, cardiomyocytes seeded in glass slides washed, fixed, and permeabilized. Then, the cells were immunolabelled with specific primary antibodies (CaSR and calnexin at 1:50 ratio) overnight at 4 °C. After rinsing, the cells were incubated with corresponding fluorescent-conjugated secondary antibody (goat anti-mouse IgG and goat anti-Rabbit IgG; Rockland Immunochemicals Inc, Limerick, PA) plus DAPI nuclear counterstain or Wheat Germ Agglutinin cytomembranes counterstain, and then observed using a microscope with fluorescence objectives (Olympus I×51, Center Valley, PA).
8
Protein Expression Analysis by Western Blot
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Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM 2-mercaptoethanol, 2% [w/v] SDS, 10% glycerol). Proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking, membranes were incubated with primary antibodies against type I collagen, DVL2, phosphorylated β-catenin (Y86), glycogen synthase kinase-3β (GSK-3β) and GAPDH (Abcam, Cambridge, MA, USA) overnight at 4°C, followed by secondary goat anti-rabbit IgG (Rockland, Limerick, PA, USA) at 37°C for 1 h. GAPDH served as an internal control.
9
Western Blot Protein Quantification
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Protein levels were analyzed by western blot as described previously (35 (link)). In brief, fifty microgram of cell lysates were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences). Blots were blocked with 5% milk-TBS for 1 h at room temperature and probed overnight at 4°C using primary antibodies against NAMPT (H-300, Santa Cruz), IDO1 (GTX54705,Genetex), and β-actin (4E8H3, Santa Cruz). Protein complexes were subsequently incubated for 1 h at room temperature with goat anti-mouse IgG (Cat# SA00001-1, Proteintech) or goat anti-rabbit IgG (Cat# 28925, Rockland) diluted at 1:2,000 in blocking buffer and then detected by Enhanced Chemiluminescence Plus (GE Healthcare). Protein bands were scanned and densitometry analysis was performed using ImageJ software.
10
Quantitative Analysis of Tubulin Modifications
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For Western blotting, protein samples that had been quantified by the bicinchoninic acid method were separated using SDS–PAGE. After transfer to a polyvinylidene fluoride membrane (Millipore), the membrane was blocked with 5% nonfat dried milk in Tris-buffered saline (TBS; pH 7.4) and incubated overnight with the primary antibodies anti-α-tubulin (1:2000; ab18251, rabbit, polyclonal; Abcam, Cambridge, United Kingdom), tyrosinated tubulin (YL1/2) rat monoclonal antibody (1:2000; ab6160, rat, monoclonal; Abcam), anti–detyrosinated tubulin (AA12; 1:2000; ab24622, mouse, monoclonal, Abcam), anti–acetylated tubulin (6-11B-1; 1:2000; ab14610, mouse, monoclonal; Abcam), and anti-Fign antibody (1:400; sc-68343, rabbit, polyclonal; Santa Cruz Biotechnology) at 4°C. After washing with TBST (TBS with 0.1% Tween 20), IRDye-800–conjugated, affinity-purified goat anti-mouse immunoglobulin G (IgG; 1:3000; Rockland, Philadelphia, PA), goat anti-rabbit IgG (1:3000; Rockland), or goat anti-rat IgG (1:3000; Rockland) antibodies were used at room temperature for 2 h. The blot was covered with x-ray film and imaged using a scanner, and the data were analyzed with PDQuest 7.2.0 software (Bio-Rad). For normalization and relative quantitative analysis of target proteins expression, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or α-tubulin was used as the control protein.
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