Whole-cell protein lysate preparation was performed with RIPA buffer (15 mM HEPES, 150 mM NaCl, 10 mM EGTA, 2% Triton X-100) supplemented with protease and phosphatase inhibitors (complete mini EDTA-free and PhosSTOP, Roche) as described before [14 (link),21 (link),35 ]. Bicinchoninic acid assay (Santa Cruz Biotechnology, Texas, USA) was used for protein quantitation according to the manufacturer's protocol. Laemmli buffer was mixed with cell lysates, denatured for 5 min at 95 °C and loaded in a 10% polyacrylamide gel (Thermo Fisher Scientific). Lysates were separated by electrophoresis at 120 V and transferred to a PVDF membrane. Membranes were blocked in 5% nonfat dry milk in TBS-T and incubated in primary antibody in 5% nonfat dry milk in TBS-T overnight at 4 °C. Antibodies and dilutions are listed in Supplementary Table 1 . Membranes were washed three times and incubated with the secondary antibody. Proteins were visualized with ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and the Fusion FX7 imaging system was used for detection (Vilber Lourmat).
Immunocruz western blotting luminol reagent
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The ImmunoCruz Western Blotting Luminol Reagent is a chemiluminescent substrate used in western blotting assays to detect the presence of target proteins. The reagent produces a luminescent signal when it reacts with the horseradish peroxidase (HRP) enzyme, which is commonly conjugated to secondary antibodies in western blotting procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Fusion fx7 imaging system, by Vilber (2 mentions)
Pierce co ip kit, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Mouse anti actin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti sam68, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Santa Cruz Biotechnology (1 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free and phosstop, by Roche (1 mentions)
Actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
Cd44 3578s, by Cell Signaling Technology (1 mentions)
Zeb1 sc 25388, by Santa Cruz Biotechnology (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Nupage novex gel, by Thermo Fisher Scientific (1 mentions)
18 protocols using immunocruz western blotting luminol reagent
1
Whole-Cell Protein Lysate Preparation and Western Blot Analysis
2
Western Blot Analysis of EMT Markers
3
Protein Isolation and Analysis
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For protein isolation, samples from nodular, tumor and non-tumor areas were lysed in buffer containing Complete Protease Inhibitor Cocktail (Roche Applied Science, Manheim, Germany) and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany). Protein extract was obtained from cells using lysis buffer containing protease and phosphatase inhibitors. For immunoprecipitation assays, cells were lysed using the Pierce Co-IP kit (Thermo Scientific, Rockford, USA) according to the manufacturer's instructions. Proteins were resolved by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, USA), incubated with the appropriate primary and secondary antibodies and detected using the ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz, USA). Densitometry analysis was performed with ImageJ software.
4
Nuclear Protein Extraction and Western Blot Analysis
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Nuclear-enriched cellular extracts were prepared by resuspending isolated germ cells or seminiferous tubules in RSB100 buffer [10 mM Tris HCl pH 7.4; 100 mM NaCl; 2,5 mM MgCl2; 0,5 % (v/v) Triton X-100; 15 mM β-glycerophosphate; 1 mM DTT; 0,5 mM NaVO3; protease inhibitor cocktail (Sigma Aldrich); RNasin Ribonuclease inhibitor 40U/ml (Promega)]. After incubation on ice for 15 min, samples were sonicated, stratified on 30% sucrose cushion and centrifuged for 15 min at 7000 g at 4°C. For total cellular extracts, isolated germ cells were resuspended in RIPA buffer [50 mM Tris pH 7.4; 1% NP-40; 0,5% Na deoxycholate; 0,1% SDS; 150 mM NaCl; 1 mM EDTA; 1 mM DTT; 0,5 mM NaVO3; protease inhibitor cocktail (Sigma Aldrich)], incubated on ice for 20 min, briefly sonicated, and centrifuged for 20 min at 12000 g at 4°C. Protein extracts were then analyzed by Western Blot using the following primary antibodies: rat anti-RNA polymerase II subunit B1 (phospho CTD Ser-2) clone 3E1 (Millipore; dilution 1:1000); rabbit anti-HSP90α/β (dilution 1:1000), anti- RNA polymerase II N-20 (dilution 1:1000) and mouse anti-ADAM3 (dilution 1:400) (Santa Cruz). Anti-rabbit, anti-mouse (GE Healthcare) and anti-rat (Santa Cruz) HRP-linked secondary antibodies were all used at 1:10000 dilution and ECL signal developed using Immunocruz Western Blotting Luminol Reagent (Santa Cruz).
5
Western Blot Analysis of Lipin-1, P-STAT6, and STAT6
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Cells were lysed in 1× NuPage LDS sample buffer [containing 100 mM dithiothreitol (DTT; Life Technologies), 1× protease inhibitor cocktail (Thermo Fisher Scientific), 1× phosphatase inhibitor cocktail 2 (Sigma Aldrich), and 1× phosphatase inhibitor cocktail 3 (Sigma Aldrich)]. Protein concentration was determined by PeirceTM 660 nm Protein Assay (Thermo Fisher Scientific) and 20 μg of each sample was separated using 4 to 12% polyacrylamide NuPAGE Novex gel (Invitrogen) run at 200 V for 55 min. Semidry transfer (Novex, SD1000) was performed for 45 min at 20 V onto a polyvinylidene difluoride (Immobilon-FL) membrane (EMD Millipore). The membranes were further blocked for 1 h at room temperature using Li-Cor blocking buffer (Li-Cor Biosciences) and incubated overnight with primary antibodies for Lipin-1 (CST #14906), P-STAT6 (CST #56554), STAT6 (CST #5397), and GAPDH (CST #2118). Goat anti-rabbit HRP secondary antibody (Jackson #111-035-144) was added to the membranes and incubated for 2 h at room temperature. Membranes were washed three times with tris buffered saline with tween 20 and incubated in ImmunoCruz Western blotting luminol reagent (Santa Cruz, sc-2048) for 1 min. Images were captured using an Amersham Imager 680 (GE Healthcare Bio-Sciences). Densitometry was performed using IQTL 8.1 (GE Healthcare Biosciences). Bands of interest were normalized to GAPDH.
6
Western Blot Protein Expression Analysis
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Whole-protein extracts were analyzed using the Immuno Cruz Western blotting Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) or the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific). The following antibodies were used: anti-GAPDH, anti-phospho-EGFR (Y1068), anti-phospho-AKT (S473), anti-phospho-p44/42 MAP kinase (T202/Y204), anti-AKT, anti-p44/42 MAP kinase, and anti-Snail (Cell Signaling Technology, Beverly, MA, USA); anti-EGFR, anti-E-cadherin, and anti-N-cadherin (Santa Cruz Biotechnology, Heidelberg, Germany); anti-vimentin antibody (DAKO, Milan, Italy); anti-α-tubulin (Sigma Aldrich, Milan, Italy). Densitometric analysis of the blots was performed using the ImageJ software v.1.8.0. Original blots were shown in Figures S5 and S6 .
7
Protein Extraction and Western Blotting
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Protein extracts were obtained from tissues or cells lysed in buffer containing Complete Protease Inhibitor Cocktail (Roche Applied Science, Germany). For immunoprecipitation assays, cells were lysed using the Pierce Co-IP kit (Thermo Scientific, USA) according to the manufacturer's instructions. Proteins were resolved by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, USA), incubated with the appropriate primary and secondary antibodies and detected using the ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz, USA).
8
Western Blot Protein Detection Protocol
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Whole-cell protein lysates were prepared by lysing cells in 15 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L EGTA, and 2% (v/v) Triton X-100 supplemented with cOmplete (Roche) and PhosStop (Roche) phosphatase inhibitors. Protein concentrations were assessed by bicinchoninic acid assay (BCA, Santa Cruz Biotechnology). For 5 minutes, 10 μg of protein was denatured in Laemmli buffer at 90°C. Samples were run on NuPage 10% polyacrylamide, 1 mm Tris-Glycine Protein Gels (Thermo Fisher Scientific), and transferred to PVDF membranes (Roche). Membranes were blocked with 5% dry milk or 5% BSA (Roth) in TBS with 0.1% (v/v) Tween-20 (Carl Roth). Membranes were probed with primary antibodies overnight at 4°C and then with secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature. Chemiluminescent detection of proteins was carried out using Immunocruz Western blotting luminol reagent (Santa Cruz Biotechnology) and the Fusion FX7 imaging system (Vilber Lourmat). Densitometry was performed using ImageJ (NIH).
9
Western Blot Analysis of Cellular Proteins
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Total proteins were extracted from cells or murine tissues by using a lysis buffer (150 mM NaCl, 15 mM MgCl2, 15 mM EGTA, 50 mM HEPES pH 7.4, 20 mM β-glycerophosphate, 10% glycerol, 1% Triton X-100) supplemented with 1% protease inhibitor cocktail (Sigma Aldrich), 0.5 mM Na3VO4, 1 mM DTT. Extracted proteins were resolved on 10% SDS-PAGE gels (10–20 μg/lane) and transferred onto nitrocellulose membrane (Amersham). Blots were incubated with the indicated primary antibody in 5% non-fat dry milk in PBS plus 0.1% Tween-20 overnight at 4°C. The primary antibodies used (1:1000) were: rat anti-RNA polymerase II (phospho-CTD Ser-2) (Millipore), mouse anti-RNA polymerase II (N-20), rabbit anti-DHX9, mouse anti-GEMIN 2, mouse anti-ACTIN, rabbit anti-Sam68, mouse anti-hnRNP F/H and goat anti-TIA-1 (all from Santa Cruz Biotechnology, Inc.), mouse anti-SMN (BD Biosciences). Detection was achieved using anti-rabbit, anti-mouse (GE Healthcare) and anti-rat (Santa Cruz Biotechnology, Inc.) HRP-linked secondary antibodies (1:10 000) and visualized by Immunocruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.).
10
SDS-PAGE and Western Blot Analysis
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blots were performed according to standard procedures. The cells were lysed in lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.6 % octylphenoxypolyethoxyethanol (IGEPAL CA-630), 1 mM EDTA] and separated on a 7.5 or 10 % Tris–glycine gel, and then transferred to a nitrocellulose membrane. Blots were blocked with 5 % skim milk in PBS and incubated with a primary antibody and washed three times with PBS-T. Next, the blots were incubated with peroxidase-labeled horse anti-mouse IgG (H + L) or peroxidase-labeled goat anti-rabbit IgG (H + L) (Vector Laboratories) and washed with three changes of PBS-T. They were then visualized by using ImmunoCruz™ Western Blotting Luminol Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Images were captured with a cooled CCD camera.
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