COS-7 cells were cultivated in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in an incubator with 5% CO2. Cells were seeded in 6-well plates at a density of 2 × 105 cells/well, and transfected with pEBMulti-Neo-SC4MOL plasmids, using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's instruction. At 48 h after the transfection, cells were harvested and lysed using protein lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% SDS, 1% sodium cholate, 1% Triton X. Proteins separated by SDS-PAGE were electrically transferred onto a PVDF membrane (GE Healthcare UK Ltd, Buckinghamshire, England). The membranes were incubated with mouse anti-His-Tag antibody (1:1000) (MBL Co., Ltd., Nagoya, Japan) at 4°C overnight. The membranes were reacted with secondary rabbit anti-mouse IgG HRP-linked antibodies (1:2000) (Cell Signalling Technology, Inc., Danvers, Massachusetts, USA) and visualized using an ECL prime Western blotting reagent (GE Healthcare UK Ltd., Buckinghamshire, England). Chemiluminescence signals were detected using the Fujifilm LAS-1000 imager (Fuji Photo Film Company, Tokyo, Japan).
Anti mouse igg hrp linked antibodies
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-mouse IgG HRP-linked antibodies are secondary antibodies that recognize and bind to mouse primary antibodies. They are conjugated with horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric reaction, allowing for the detection and visualization of target proteins in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit, by Cell Signaling Technology (2 mentions)
Anti rabbit igg horseradish peroxidase hrp linked antibodies, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti gfp, by Roche (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ecl prime western blotting reagent, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Formic acid, by Fujifilm (1 mentions)
Glycyrrhizic acid, by ChemFaces (1 mentions)
Ginsenoside rb1, by ChemFaces (1 mentions)
Saikosaponin a, by ChemFaces (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Methanol, by Duksan Pure Chemicals (1 mentions)
Ethanol, by Duksan Pure Chemicals (1 mentions)
11 protocols using anti mouse igg hrp linked antibodies
1
Heterologous Protein Expression in COS-7 Cells
2
Phytochemical Quantification and Inflammatory Signaling
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For sample extraction, ethanol, methanol, and ethyl acetate (extra-pure-grade) were purchased from Duksan Pure Chemicals Co. (Ansan, Republic of Korea). For high-performance liquid chromatography (HPLC) quantification, HPLC-grade acetonitrile, methanol, and water were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Formic acid (99%, HPLC-grade) was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan), and acetic acid (99%) and LPS (from Escherichia coli O111:B4, L4391) were provided by Sigma-Aldrich (St. Louis, MO, USA). Glycyrrhizic acid (CAS No. 1405-86-3, CFN99151, CFS202002, purity 99.5%), ginsenoside Rb1 (CAS No. 41753-43-9, CFN99964, CFS202101, purity 98.9%), baicalin (CAS No. 21967-47-9, CFN99111, CFS202003, purity 98.0%), saikosaponin A (CAS no. 20736-09-8, CFN99987, CFS202102, purity 99.4%), and saikosaponin B2 (CAS No. 58316-41-9, CFN99126, CFS202102, purity 99.7%) were supplied by Chemfaces (Wuhan, China). Antibodies against NOS2 (iNOS), COX-2, p38, phosphorylated-p38 (pp38), ERK, phosphorylated-ERK (pERK), JNK, phosphorylated-JNK (pJNK), NRF2, β-actin, anti-rabbit and anti-mouse IgG HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
DENV Protein Detection by Dot Blot
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Fractions from each sucrose gradient (200 µl) were applied to a nitrocellulose membrane (Amersham Biosciences) with a dot blot apparatus. The membrane was blocked in 5% w/v skim milk in PBST for 1 h at room temperature before being incubated in rabbit anti-DENV E polyclonal antibodies (GeneTex) and rabbit anti-DENV C polyclonal antibodies (Novusbio) overnight at 4 °C. Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies or anti-mouse IgG HRP-linked antibodies (Cell Signaling Technology).
4
Western Blot Analysis of Inflamed Skin
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Skin samples from the back lesions of mice were cut into pieces, snap-frozen in liquid nitrogen, and stored at −80°C until use. The samples were homogenized in T-PER buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail and a phosphatase inhibitor cocktail. Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) membranes. The blots were blocked and incubated at 4°C overnight with antibodies against p-IκB and β-actin (Cell signaling, Beverly, MA, USA). The membranes were then incubated with anti-rabbit IgG or anti-mouse IgG HRP-linked antibodies (cell signaling). Chemiluminescence was measured by using the chemiDoc system (Bio-rad) and analyzed with Quantity One software (Bio-Rad).
5
Linaclotide-Induced CFTR Activation
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Linaclotide was provided by Ironwood Pharmaceuticals Inc (Cambridge, MA). EZ‐Link Sulfo‐NHS‐SS‐Biotin and streptavidin‐agarose resins were obtained from Thermo Scientific (Rockford, IL). 8‐Bromoguanosine 3′,5′‐cyclic monophosphate sodium salt monohydrate (8Br‐cGMP), bumetanide, GlyH‐101, and NKH477 were obtained from Sigma‐Aldrich (St. Louis, MO). The PKA inhibitor H7 and PKG inhibitor H8 dihydrochloride were purchased from AbCam (Cambridge, MA). The mouse monoclonal CFTR‐450 antibody raised against full‐length human CFTR was obtained from Cystic Fibrosis Foundation Therapeutics, University of North Carolina‐Chapel Hill. The affinity‐purified polyclonal antibody AME4991 raised against rat CFTR was generated by Dr. Ameen (Ameen et al. 2000 ; Golin‐Bisello et al. 2005 ; Jakab et al. 2011 ). Phospho‐VASP (Ser239) and antimouse IgG, HRP‐linked antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA). The cGKII (PRKG2) rabbit polyclonal antibody was obtained from Proteintech Group Inc. (Chicago, IL) and β‐actin monoclonal antibody from Sigma‐Aldrich (St. Louis, MO). All other drugs, reagents, and chemicals were obtained from Sigma‐Aldrich unless otherwise stated.
6
Immunoblotting Analysis of DENV Proteins
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Cell were lysed at 4 °C for 30 min with lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% [v/v] Triton X-100; protease inhibitor cocktail [Roche]). Cell lysates were centrifuged at 12,000 × g for 10 min and clarified supernatants were collected. The total protein concentrations were determined by the Bradford method against a bovine serum albumin standard. Protein was boiled in SDS-PAGE sample buffer (125 mM Tris–HCl, pH 6.8; 4% [v/v] SDS; 20% [v/v] glycerol; 0.004% [w/v] bromphenol blue), and separated by 10% SDS-PAGE. Gels were electro-blotted onto a polyvinylidene fluoride membrane (Pall) using a semi-dry transfer system (Bio-Rad). DENV-2 E and CA proteins were detected with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV C polyclonal antibody (Novusbio), respectively. DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS). Endogenous β-tubulin was detected with a mouse anti-β-tubulin monoclonal antibody (Sigma Aldrich). Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies or anti-mouse IgG HRP-linked antibodies (Cell Signaling Technology).
7
Immunoblotting analysis of NF-κB and JNK signaling
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THP-1 cells were rested overnight and then treated with 5 μM #1 in the presence or absence of 4 ng/mL LPS for indicated time periods. Cells were lysed with PhosphoSafe Extraction Reagent (EMD Millipore, Billerica, MA) supplemented with protease inhibitor cocktail (Roche, Manheim, Germany) and 0.1% SDS (Thermo Fisher Scientific). Protein concentrations were measured using Pierce BCA protein assay kit (Thermo Fisher Scientific). Ten μg of reduced and denatured protein per sample was then separated by gel electrophoresis and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat milk in 0.1% Tween-20 Tris-buffered saline (TBST) and subsequent washes were done in TBST. Primary antibody and secondary antibody were diluted in 5% bovine serum albumin- (BSA-) TBST and 5% nonfat milk-TBST. Anti-phospho NF-κB p65 (#3033), anti-NF-κB p65 (#8242), anti-IκBα (#4814), anti-phospho JNK (#9251), anti-JNK (#9252) anti-β-actin (#3700), anti-rabbit (#7074), and anti-mouse IgG HRP-linked antibodies (#7067) were all purchased from Cell Signaling Technology (Danvers, MA).
8
Western Blot Analysis of Adenovirus E1A
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The cells were homogenized with RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor mixture (Sigma-Aldrich). After being frozen and thawed, the homogenates were centrifuged at 15,000 × g at 4 °C for 10 min, and the supernatants were collected. The lysates were subjected to SDS–PAGE on 7.5% polyacrylamide gel, and then transferred onto polyvinylidene fluoride membranes (Millipore). After the reaction was blocked with 1% skim milk in TBS containing 0.1% Tween 20 at room temperature for 1 h, the membranes were incubated with mouse anti-Adenovirus-5 E1A (sc-58658, Santa Cruz Biotechnology) or mouse anti-β-Actin antibody (A5316, Sigma-Aldrich) at 4 °C overnight, followed by reaction with anti-mouse IgG, HRP-linked antibodies (Cell Signaling Technology) at room temperature for 1 h. The band was visualized by Chemi-Lumi One Super (Nakalai Tesque) and the signals were read using an LAS-4000 imaging system (FUJIFILM).
9
Western Blot Analysis of Signaling Proteins
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For western blot analysis, 1 × 106 cells were lysed with RIPA buffer (08714-04, Nakalai Tesque). SDS sample buffer was added, and the mixture was incubated at 93 °C for 3 min. The extracted proteins were separated on Bollt 4–12%, Bis-Tris, 1.0 mm, Mini Protein Gel (NW04120BOX, Thermo Fisher Scientific) and blotted onto an Immobilon-P PVDF Membrane (IPVH00010, Merck) using a Mini PROTEAN Tetra Cell (Bio-Rad). The transferred membranes were incubated with the following primary antibodies: α-tubulin (ab7291, Abcam), pSMAD1/5/9 (9511, Cell Signaling Technology), pSMAD2 (3108, Cell Signaling Technology), pMAPK (4376, Cell Signaling Technology), pSTAT3 (9131, Cell Signaling Technology) and STAT3 (564533, BD Bioscience). The primary antibodies were detected with anti-rabbit IgG, HRP-linked antibodies (7074, Cell Signaling Technology) and anti-mouse IgG, HRP-linked antibodies (7076, Cell Signaling Technology), followed by detection using ECL Prime Western Blotting Detection Reagent (RPN2236, Amersham). Chemiluminescence images were acquired using the ImageQuant LAS 4000 (GE Healthcare) and Ambersham ImageQuant 800 (Cytiva) systems. Uncropped western blot images are shown in Supplementary Figs. 2 and 3 .
10
Protein Extraction and Immunoblotting for Plant Viruses
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Total protein was isolated from infiltrated leaf patches with protein extraction buffer (containing 50 mM Tris-HCl (pH 6.8), 4.5% (m/v) SDS, 7.5% (v/v) 2-Mercaptoethanol, 9 M carbamide). Immunoblotting was performed with primary mouse polyclonal antibodies, followed by anti-mouse IgG HRP-linked antibodies (1:5000; Cell Signaling Technology, Cat#: 7076, USA); primary antibodies used are as follows: anti-GFP (1:5000; ROCHE, Cat#: 11814460001, USA), and custom-made anti-PVX CP (1:5000) and anti-TYLCV CP (1:5000)59 ,60 (link).
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