Cells were scraped, lysed, and collected in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.002% bromophenol blue). Protein concentrations were determined using a protein assay kit (Bio-Rad DC; Bio-Rad, Segrate, Italy). Cell extracts were separated on 7.5% or 10% (w/v) homogeneous slab gels (40 μg of protein/lane) using SDS-PAGE and then transferred to nitrocellulose membranes (Protran; Whatman-GE Healthcare, Milan, Italy). Membranes were hybridized with a rabbit polyclonal anti-LC3 antibody (1:1,000 dilution, Sigma-Aldrich), mouse monoclonal anti-cytochrome C1 antibody (1:10 dilution, Santa Cruz Biotechnology), mouse monoclonal anti-beta tubulin or beta actin antibody (1:1,000 dilution, Santa Cruz Biotechnology) followed by an incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1:10,000 dilution, GE Healthcare, Cologno Monzese, Italy). The relevant proteins were detected using chemiluminescence kits (Pierce EuroClone S.p.A., Pero, Italy), and the signals were acquired and analysed using an image acquisition system (Uvitec mod Alliance 9.7, Uvitec, Cambridge, UK). A mouse monoclonal anti-GAPDH antibody (1:5,000 dilution, Merck S.p.a., Vimodrone, Italy) was used as a loading control.
Horseradish peroxidase conjugated anti rabbit or anti mouse igg
Manufactured by GE Healthcare
Sourced in United Kingdom
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG is a laboratory reagent used for immunodetection applications. It consists of horseradish peroxidase enzyme covalently conjugated to antibodies that specifically recognize and bind to rabbit or mouse immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (3 mentions)
Mod alliance 9, by Uvitec (2 mentions)
Whatman, by Cytiva (2 mentions)
Protran, by Cytiva (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Immobilon, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Alexa 488 conjugated anti rabbit or alexa 594 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Ecl plus chemiluminescence system, by GE Healthcare (2 mentions)
Amersham hybond p, by GE Healthcare (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Image studio software, by LI COR (2 mentions)
Las 4000, by Cytiva (2 mentions)
Anti lc3 antibody, by Merck Group (1 mentions)
Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by GE Healthcare (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti phospho erk, by Cell Signaling Technology (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
21 protocols using horseradish peroxidase conjugated anti rabbit or anti mouse igg
1
Quantitative Western Blot Analysis
2
Immunoblotting for Protein Analysis
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Cells were scraped and collected in sample lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.002% bromophenol blue). Protein content was determined using a protein assay kit (Bio-Rad DC; Bio-Rad Laboratories Srl, Milan, Italy). Cell extracts were separated on 7.5% or 10% (w/v) homogeneous slab gels (40 μg of protein/lane) using SDS-PAGE and then transferred to nitrocellulose membranes (Protran; Whatman-GE Healthcare, Milan, Italy). Membranes were hybridized with a mouse monoclonal anti-β tubulin antibody (1 : 500 dilution, Thermo Fisher Scientific) or mouse monoclonal anti-β actin antibody (1 : 1,000 dilution, Santa Cruz Biotechnology Inc., Heidelberg, Germany) followed by an incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1 : 10,000 dilution, GE Healthcare, Cologno Monzese, Italy). The relevant proteins were detected using chemiluminescence kits (Pierce EuroClone S.p.A., Pero, Italy), and the signals were acquired and analyzed using an image acquisition system (Uvitec mod Alliance 9.7, Uvitec, Cambridge, UK). A mouse monoclonal anti-GAPDH antibody (1 : 10,000 dilution, Merck S.p.A., Vimodrone, Italy) was used as a loading control.
3
Western Blot Analysis of Akt and Erk
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The cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 125 mM NaCl, 0.1% Triton X-100, and 5 mM ethylenediaminetetraacetic acid containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer onto a polyvinylidene difluoride membrane. After blotting with the following antibodies from Cell Signaling Technology (Beverly, MA, USA) against phosphorylated Akt (Ser473, 1:500), phosphorylated Akt (Thr308, 1:500), non-phosphorylated Akt (1:1000), phosphorylated Erk1/2 (Thr202/Tyr204, 1:200), non-phosphorylated Erk1/2 (1:200), and β-actin (1:1000), horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1:1000; GE Healthcare, Little Chalfont, Buckinghamshire, UK) were used as secondary antibodies. The signals were visualized with enhanced chemiluminescence.
4
Western Blot Protein Analysis Protocol
5
Regulation of ERK Phosphorylation by RKIP
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The inhibitory effect of RKIP-TV-delRBD on ERK phosphorylation was tested by immunoblotting, as described previously (Yamamoto et al., 2012 (link)). In brief, human embryonic kidney 293T (HEK293T) cells transfected with pEGFP-RKIP-WT or pEGFP-RKIP-TV-delRBD were treated with or without TPA (100 nM) for 5 min and lysed in lysis buffer containing the following: 150 mM NaCl, 50 mM Tris, pH 8, 1 mM EDTA, 0.2% NP-40 alternative (Calbiochem), 10% glycerol, 1X proteinase inhibitor cocktail, and 1X phosphatase inhibitor cocktail. The supernatants of cell lysates were subjected to SDS-PAGE analysis to detect phosphorylated ERK, ERK, and GFP by immunoblotting. Primary antibodies used were rabbit anti-ERK (Cell Signaling Technology), rabbit anti-phospho-ERK (Cell Signaling Technology) or mouse anti-GFP (Roche Applied Science). Secondary antibodies used were horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare) antibodies.
6
SDS-PAGE and Western Blot Analysis
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Protein samples were diluted with sample buffer(125 mM Tris-HCl, 4 % SDS, 2 % 2-mercaptoethanol, 20 % glycerol, 0.01 % bromophenol blue, pH6.8) and denatured at 95 °C for 3 min. Equal amounts of samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels (5–20 %; Atto, Tokyo, Japan) in running buffer (25 mM Tris, 192 mM glycine and 0.1 % SDS), followed by Western blot, using polyvinylidene difluoride microporous membrane, blocking this membrane in blocking One (Nacalai tesque), and incubating with the primary antibodies in PBS containing 4 % BSA (Nacalai tesque) overnight at 4 °C. The membranes were then washed with 20 mM TBS containing 0.1 % Tween 20 (TBS-t) and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare, Little Chalfont, UK) in TBS-t for 1 h at room temperature. The specific reaction was visualized using the ECL method (GE Healthcare).
7
Hypoxia-Induced Protein Analysis
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Protein analysis was performed by immunoblotting as described previously [89 (link)]. Briefly, LNCaP or 22Rv1 cells were incubated under hypoxic conditions (1% O2) for 24 h or up to 7 days as indicated. Cells were harvested and the protein concentration estimated by the Bradford assay. Equal amounts of proteins from the samples were resolved on a SDS-acrylamide gel then transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were probed with primary antibodies and the detection was done using horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG (GE Healthcare,) and ECL Prime detection kit (GE Healthcare).
8
Western Blot Protein Analysis Protocol
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Whole-cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 50 mM NaF, 0.1% SDS, 1% Triton-100, and Protease Inhibitor Cocktail). Protein samples (15 μg) were separated by electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose membranes (Immobilon; Millipore, Bedford, MA). Membranes were blocked with 5% skim milk at room temperature for 1 h before incubation with primary antibodies at 4°C for 12 h. The membranes were washed 4 times in PBS containing Tween-20, incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare UK Ltd., Buckinghamshire, UK) at room temperature for 1 h, and again washed 4 times in PBS-Tween 20. Protein bands were detected using the ECL Plus Western Blotting Detection System (GE Healthcare).
9
Protein Extraction and Western Blotting
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Proteins were extracted using standard RIPA lysis buffer containing freshly added protease and phosphatase inhibitors. Frozen tumors were grinded and homogenized prior to lysis. Western blotting was performed using standard protocols. Briefly, membranes were blocked for 1 h at RT in 5% milk (BioRad) and incubated overnight at 4 °C with the indicated antibody dilutions prepared as recommended by manufacturer. After washing in PBS containing 0.1% Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare). Signal was developed using Supersignal WestPico PLUS (Pierce) and films scanned as digital images. Adobe Photoshop software was used for quantification of digital images.
10
Immunoblotting Assay for Epithelial-Mesenchymal Transition
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Lysates for immunoblotting were prepared from frozen cell pellets or snap-frozen xenograft tumors using RIPA buffer (Santa Cruz Biotechnology) with 1% Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentrations were determined using Bradford methodology (Bio-Rad). Proteins (50 μg per load) were resolved onto a 4–20% SDS-PAGE gradient gel (Bio-Rad), transferred onto a nitrocellulose membrane, blocked with 5% milk for 2 hrs, then incubated in primary antibody at 4 °C overnight (ZEB1 H-102, 1:200, sc-25388; ZEB2 H-260, 1:200, sc-48789; E-cadherin H-108, 1:500, sc-7870; N-cadherin, 1:500, ab18203; Vimentin V9, 1:200, sc-6260; β-Actin AC-15 1:10,000, sc-69879). All primary antibodies were obtained from Santa Cruz Biotechnology except for the anti N-cadherin antibody, which was obtained from AbCam. Membranes were then washed in TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5000, GE Healthcare) for 1 h at room temperature. Blots were developed and visualized using the Amersham™ ECL™ Western Blotting Detection Reagent kit (GE Healthcare Life Sciences). Antibody for β-actin was used as a loading control.
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