Cells were lysed using RIPA buffer and analysed by western blotting as described56 (link). Primary antibodies used were: pJAK1(Tyr1034/1035; 74129T, 1:500 dilution), pJAK1(Tyr1022/1023; 3331S, 1:500 dilution), pSTAT1(Tyr701; 7649T, 1:500 dilution), pSTAT3(Ser727; 9134T, 1:500 dilution) from Cell Signaling and ERK2 (sc-1647) from Santa Cruz Biotechnology. Detection was through enhanced chemiluminescence ECL using horseradish peroxidase-coupled secondary antibodies (1:2,000 dilution; GE Healthcare) and NOVEX ECL Chemi Substrate (Thermo Fisher).
Horseradish peroxidase coupled secondary antibody
Manufactured by GE Healthcare
Sourced in United States
Horseradish peroxidase-coupled secondary antibodies are laboratory reagents used in various immunoassay techniques. They consist of secondary antibodies conjugated with the enzyme horseradish peroxidase. These antibodies can bind to primary antibodies and catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (2 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P jak1, by Cell Signaling Technology (1 mentions)
Erk2 sc 1647, by Santa Cruz Biotechnology (1 mentions)
Novex ecl chemi substrate, by Thermo Fisher Scientific (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Polyvinyl difluoride membrane, by GE Healthcare (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Pepstatin, by Merck Group (1 mentions)
Anti bcl xl, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Calpain inhibitor, by Merck Group (1 mentions)
Sodium orthovanadate, by Fujifilm (1 mentions)
Anti mdr1, by Santa Cruz Biotechnology (1 mentions)
Anti survivin, by Santa Cruz Biotechnology (1 mentions)
13 protocols using horseradish peroxidase coupled secondary antibody
1
Western Blotting of JAK-STAT Signaling
2
Cytoplasmic Protein Extraction and Analysis
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Cytoplasmic cell fraction was collected by using cell lysis buffer (20 mM Tris-HCl (pH 8.0; Wako), 2 mM EDTA (Wako), 0.5% NP-40 (Wako), 1 μM pepstatin (Sigma), 1 μM leupeptin (Sigma), 2 mM sodium orthovanadate (Wako), 1 μM calpain inhibitor (Sigma), phosphatase inhibitor cocktail I/II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). The protein contained amount of these fraction was evaluated using a bicinchoninic acid protein-assay kit (Wako). The extracts (40 μg of protein) were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinyl difluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were reacted with the following antibodies: anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-MDR1, anti-BCRP, anti-MRP1, anti-LRP1 (Santa Cruz Biotechnologies, CA, USA), anti-phospho-Src (Tyr527), anti-Src (Cell Signaling Technology, Beverly, MA), and anti-β-actin (Sigma) as an internal control. The membranes were reacted with horseradish peroxidase-coupled secondary antibodies (GE Healthcare) for 1 h at room temperature and proteins were assessed using a Luminata Forte (Merck Millipore, Nottingham, UK).
3
Western Blot Analysis of Protein Samples
4
Western Blot Quantification Protocol
5
Cell Lysis and Protein Immunoblotting
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Cells were routinely lysed in buffer composed of 50 mM Tris–HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 1 tablet protease inhibitor (ROCHE) per 10 ml. Following lysis, cells were incubated on ice for 10 min before centrifugation at 12,500 rpm for 10 min at 4 °C. Protein concentration was determined using BCA assay, and then equal amounts of protein per condition were diluted in 4× sample buffer (BIO-RAD) supplemented with β-mercaptoethanol. Samples were then loaded onto pre-cast PROTEAN any kDa polyacrylamide gels (BIO-RAD). Following migration, proteins were transferred onto a nitrocellulose membrane, and subjected to standard immunoblotting (briefly, blocking in Tris-buffered saline 0.1% Tween-20 (TTBS), 5% milk for 1 h, followed by overnight incubation in primary antibody diluted in TTBS 5% bovine serum albumin (BSA), then 3× TTBS 0.1%Tween washes, incubation with horseradish peroxidase-coupled secondary antibodies (GE HEALTHCARE) diluted 1/10,000 in TTBS 5% milk for 1 h at room temperature followed by a final three washes). Proteins were revealed using ECL and Fusion chemiluminescence imaging system. Protein band intensity was quantified using BioID software.
6
Western Blot Analysis of CD8+ T Cell Proteins
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Stimulated CD8+ T cells were lysed in 1× RIPA buffer and proteins extracted by sonication.
Protein concentration was determined using the Pierce™ BCA™ Protein Assay Kit (Thermo Fisher). Equivalent amounts of proteins were denatured in Laemmli buffer under reducing conditions, separated by 7%–10% SDS‐PAGE and transferred to PVDF‐membrane using a transblot electrophoresis transfer cell (Fisherbrand). Membranes were blocked with 5% skim milk powder in Tris–HCl‐buffered saline/0.1% Tween (TBST) for 1 h, incubated overnight with primary antibodies against perforin (Invitrogen), granzyme B (Cell Signaling), and GAPDH (Cell Signaling), washed in TBST and incubated with horseradish peroxidase‐coupled secondary antibodies (GE Healthcare) for 1 h. For protein detection, an enhanced chemiluminescence detection reagent (BioRad) was used. Densitometric quantification was done with Quantity one software (Bio‐Rad).
Protein concentration was determined using the Pierce™ BCA™ Protein Assay Kit (Thermo Fisher). Equivalent amounts of proteins were denatured in Laemmli buffer under reducing conditions, separated by 7%–10% SDS‐PAGE and transferred to PVDF‐membrane using a transblot electrophoresis transfer cell (Fisherbrand). Membranes were blocked with 5% skim milk powder in Tris–HCl‐buffered saline/0.1% Tween (TBST) for 1 h, incubated overnight with primary antibodies against perforin (Invitrogen), granzyme B (Cell Signaling), and GAPDH (Cell Signaling), washed in TBST and incubated with horseradish peroxidase‐coupled secondary antibodies (GE Healthcare) for 1 h. For protein detection, an enhanced chemiluminescence detection reagent (BioRad) was used. Densitometric quantification was done with Quantity one software (Bio‐Rad).
7
Western Blot Analysis of STAT4 and pSTAT4
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Total splenic leukocytes and FACS-sorted CD8, LCMV-specific (LCMV-Tet+) CD8, and nonspecific (LCMV-Tet−) CD8 T cells were prepared and lysed for Western blot analysis as previous described (11 (link)). Mouse monoclonal antibodies against STAT4 (clone 8) and pSTAT4 (clone 38) were purchased from BD Biosciences. A rabbit anti-pSTAT4 polyclonal antibody was also used. A rabbit anti-β-actin polyclonal antibody was obtained from Abcam (Cambridge, United Kingdom). Proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and then incubated with antibodies. Reactive bands on Western blots were detected with horseradish peroxidase-coupled secondary antibodies and an enhanced chemiluminescence detection system (GE Healthcare). Anti-STAT4 and -pSTAT4 antibodies were loaded. A rabbit anti-β-actin polyclonal antibody from Abcam was used as a loading control. Control samples were prepared from STAT4-deficient (9 (link)) and STAT1-deficient (50 (link)) mice.
8
Immunoblotting for FIAT in Osteoblasts
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Proteins were extracted from primary osteoblasts cultures in radioimmunoprecipitation assay buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). For detection of FIAT, a polyclonal rabbit antibody directed against the N-terminus of FIAT was used (15 (link)). As a loading control, a monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO) was used. Horseradish peroxidase-coupled secondary antibodies (GE Healthcare) and ECL reagent (GE Healthcare) were used for detection.
9
Western Blot Analysis of Total Cell Lysates
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To prepare the total cell lysate, cells grown in 60 mm plate were washed twice with PBS and solubilized on ice for 30 min with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% SDS, 1% Nonidet P40, 1% sodium deoxycholate; ThermoFisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail tablets and protein phosphatases inhibitor cocktails (Roche, Indianapolis, IN, USA). Protein concentration was then determined using a BCA assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were applied and separated on 10 and 12% SDS-PAGE under reducing conditions. The proteins were then transferred to a PVDF membrane and the membrane was blocked. The membrane was then incubated with primary antibodies, and proteins were detected using horseradish peroxidase-coupled secondary antibodies (GE Healthcare Life Science, Piscataway, NJ, USA). Bound antibodies were visualized with ECL Plus or ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Science). Images were acquired using an ImageQuant LAS 4000 system (GE Healthcare Life Science) and densitometric analysis was performed using ImageJ v1.52c (National Institutes of Health, MD, USA). The brightness and contrast of the images were adjusted in ImageJ (National Institutes of Health) and Photoshop v7.0.1 (Adobe, San Jose, CA, USA).
10
Preparation and Analysis of Cell Lysates
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For preparation of total cell lysate, cells grown in a six-well plate were washed twice with PBS and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH 7.5, 300 mM NaCl, 2% (wt/vol) CHAPS, protease inhibitor cocktail (Roche, Indianapolis, IN) on ice for 30 min and then protein concentrations were determined. Proteins precipitated with trichroloacetic acid were lysed with NuPAGE LDS sample buffer (Invitrogen) supplemented with 80 mM dithiothreitol. The appropriate amounts of proteins were applied and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen) with MES or MOPS SDS running buffer (Invitrogen). After transfer to PVDF membrane, blocking and incubation with primary antibodies, proteins were detected using horseradish peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ) and ECL Plus or ECL Prime western blotting detection reagents (GE Healthcare Life Sciences).
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