Protein extraction was performed using ice-cold RIPA buffer. Bradford assay (Bio-Rad) was used to assess protein concentration in the extracts. Proteins were resolved by electrophoresis in SDS-polyacrylamide gels (10%). Then, they were transferred to a nitrocellulose membrane (Macherey-Nagel, Düren Germany). The membrane was blocked for 1 h, at room temperature, in PBST with 5% nonfat milk and then with 5% bovine serum albumin incubated with primary antibodies overnight at 4 °C. Anti-PXR (1:500, sc-48403, Biotechnology Inc., Santa Cruz, CA, USA) and anti-b-Actin (1:1000 sc-8035; Biotechnology Inc., Santa Cruz, CA, USA) were used as primary antibodies. Horseradish peroxidase-conjugated secondary antibodies (Dako, CA, USA) were used at 1:5000 dilution. The detection of the immunoreactive bands was performed with the LumiSensor Chemiluminescent HRP Substrate kit (GenScript, NJ, USA). Relative protein amounts were evaluated by a densitometry analysis using ImageJ software. We used three independent samples for each group and β-actin for the normalization [40 (link)].
Lumisensor chemiluminescent hrp substrate kit
Manufactured by GenScript
Sourced in United States
The LumiSensor Chemiluminescent HRP Substrate kit is a laboratory product designed to detect and quantify horseradish peroxidase (HRP) in various applications. The kit provides a chemiluminescent substrate that emits light upon reaction with HRP, which can be measured using a luminometer or other light detection equipment.
Automatically generated - may contain errors
Lab products found in correlation
Porablot ncp, by Macherey-Nagel (3 mentions)
Superrx fuji medical x ray film, by Fujifilm (3 mentions)
Mini protean tetra cell, by Bio-Rad (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Hybond p hydrophobic polyvinylidene difluoride pvdf membranes, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Macherey-Nagel (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab45939, by Fortrea (1 mentions)
Prb 155p, by Abcam (1 mentions)
Secondary goat anti mouse igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Anti actin c4, by MP Biomedicals (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti gfp, by Roche (1 mentions)
Gelcode blue stain reagent, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Criterion tris hcl precast gel, by Bio-Rad (1 mentions)
19 protocols using lumisensor chemiluminescent hrp substrate kit
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Cytoskeletal Proteins
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Cells were lysed in RIPA buffer, and protein concentration was determined with the Bio-Rad Protein assay using protein assay dye reagent concentrate (Bio-Rad, cat. #500-0006). Total lysates were separated on a 10 % SDS-PAGE gel for 1 h at 200 V. Proteins were subsequently transferred to a nitrocellulose membrane via semi-transfer for 25 min at 20 V, blocked with 5 % milk in TBS-Tween for 1 h at room temperature, and then incubated with the appropriate primary antibody at 4 °C overnight. Primary antibodies used were: rabbit polyclonal to vimentin (Abcam, cat. #ab45939), rabbit polyclonal to keratin 14 (Covance, cat. #PRB-155P), and rabbit polyclonal to actin (Abcam, cat. #ab8226). Membranes were then incubated in blocking buffer plus anti-rabbit HRP-conjugated secondary antibody (Santa Cruz, cat. #SC2054) for 1 h at room temperature and exposed using the LumiSensor™ chemiluminescent HRP substrate kit (GenScript, cat. #L00221V500).
3
Western Blot Quantification of PC1 and p-RUNX2
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Protein extracts were resolved by electrophoresis in SDS‐polyacrylamide gels with varying densities (6% for PC1; 10% for p‐RUNX2) and transferred onto a nitrocellulose membrane (Porablot NCP, Macherey‐Nagel, Duren, Germany). Membranes were incubated overnight at 4°C with the primary antibodies [dilutions were 1:250 for antibodies against PC1 and 1:1000 for antibodies against p‐RUNX2 and actin in PBS‐0.1% Tween 20® detergent (PBST) containing 1% non‐fat milk]. Detection of the immunoreactive bands was performed with the LumiSensor Chemiluminescent HRP Substrate kit (GenScript). Relative protein amounts were evaluated by densitometric analysis using ImageJ software and normalized to the corresponding actin levels.
4
Phosphoinositide Binding Assay
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A phosphoinositide array membrane (PIP Strip, Invitrogen) was used per manufacturer’s instructions to qualitatively assess the affinities of His-tagged PlyCB and selected mutants to phospholipids. To detect bound proteins that interact with specific phospholipids, mouse anti-His mAbs followed by goat anti-mouse IgG [HRP] were used and the blot was developed with the LumiSensor Chemiluminescent HRP Substrate Kit (all from GenScript, Piscataway, NJ).
5
Quantitative Western Blot Analysis
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Cultures for total protein extraction were grown in MM supplemented with urea at 25°C for 16 h. Total protein extraction was performed as previously described (Galanopoulou et al., 2014) . Equal sample loading was estimated by Bradford assays and Coomasie staining. Total proteins (30 μg) were separated by SDS-PAGE (10% w/v polyacrylamide gel) and electroblotted (Mini PROTEAN™ Tetra Cell, BIORAD) onto PVDF membranes (Macherey-Nagel, Lab Supplies Scientific SA, Hellas) for immunodetection. The membrane was treated with 2% (w/v) non-fat dried milk, and immunodetection was performed with a primary mouse anti-GFP monoclonal antibody (Roche Diagnostics, Hellas), a mouse anti-actin monoclonal (C4) antibody (MP Biomedicals Europe) and a secondary goat anti-mouse IgG HRP-linked antibody (Cell Signaling Technology Inc, Bioline Scientific SA, Hellas). Blots were developed by the chemiluminescent method using the LumiSensor Chemiluminescent HRP Substrate kit (Genscript USA, Lab Supplies Scientific SA, Hellas) and SuperRX Fuji medical X-Ray films (FujiFILM Europe, Lab Supplies Scientific SA, Hellas). Densitometry analysis of Fur-GFP, free-GFP and actin-specific bands was performed using the ImageJ software (Schneider et al., 2012) . The relative density of the Fur-GFP and free-GFP bands were quantified and normalised by dividing with the respective actin-band density.
6
Immunoblotting of Fungal Proteins
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Total protein extraction was performed using dry mycelia from cultures grown in minimal media supplemented with NaNO3 at 25 °C, as previously described [44 (link)]. Total proteins (25–50 μg, estimated by Bradford assays) were separated in 8–10 % (w/v) polyacrylamide gels. Immunodetection was performed on PVDF membranes (GE Healthcare Life Sciences, Amersham, United Kingdom) using primary anti-FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA), anti-GFP (Roche Diagnostics, Basel, Switzerland), anti-actin (C4) (MP Biomedicals, Santa Ana, CA, USA) and an HRP-linked secondary antibody (Cell Signaling Technology Inc, Danvers, MA, USA). Blots were developed using the LumiSensor Chemiluminescent HRP Substrate kit (Genscript, Piscataway, NJ, USA) and SuperRX Fuji medical X-ray films (FujiFILM, Minato City, Tokyo, Japan).
7
Western Blot Analysis of Protein Targets
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Protein extracts were resolved by electrophoresis in SDS‐polyacrylamide gels with varying densities (6% for PC1; 8% for mTOR and p‐mTOR; 10% for p70S6K, p‐p70S6K, AKT, p‐AKT and PTEN; 15% for p‐4EBP1) and transferred to a nitrocellulose membrane (Porablot NCP, Macherey‐Nagel). Membranes were incubated overnight at 4°C with the primary antibodies (dilutions were 1:250 for antibodies against PC1, PC2, mTOR, p70S6K, p‐p70S6K; 1:1000 for p‐mTOR, AKT, p‐AKT, PTEN, p‐4EBP1, actin in PBST containing 1% non‐fat milk). Detection of the immunoreactive bands was performed with the LumiSensor Chemiluminescent HRP Substrate kit (GenScript). Relative protein amounts were evaluated by densitometric analysis using Image J software and normalized to the corresponding actin levels. The experiments were performed in triplicate.
8
Protein Characterization via Immunoblotting
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Protein samples were combined with Laemmli sample buffer and then loaded on 10% Criterion™ Tris–HCl Precast Gels (from Bio-Rad) and run at a constant voltage. Coomassie staining was achieved using GelCode® Blue Stain Reagent (from Thermo Scientific). For immunoblot, proteins were transferred to nitrocellulose membrane using a Trans-Blot® SD Semi-Dry Transfer Cell system (from Bio-Rad). Specific proteins were detected using peptide antibodies generated by Thermo Scientific’s Pierce Custom Antibody Services: CDH-1 (NCU00206), GH5-1 (NCU00762), GH10-1 (NCU05924), GH61-5 (NCU08760), GH51-1 (NCU02343) and GH10-2 (NCU08189). In addition, an antibody directed against GLA-1 (NCU01517) was kindly provided by Stephen Free. These primary antibodies were detected using a goat anti-rabbit IgG (whole molecule)-peroxidase antibody (from Sigma) secondary antibody. The specific protein bands were visualized using the LumiSensor™ Chemiluminescent HRP Substrate Kit (from GenScript) and ChemiDoc™ XRS+ molecular imager with Image Lab™ software (from Bio-Rad).
9
Western Blot Analysis of Protein Expression
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Harvested cells were lysed with RIPA solution (50 mM Tris-HCl, 1% NP-40, 0.25% Na-Deoxycholate, 150 mM NaCl, 1 mM EDTA with protease and phosphatase inhibitors). Equal amounts of protein were subjected to SDS-PAGE using 10% polyacrylamide gels under reducing conditions. Separated protein bands were transferred to nitrocellulose membranes in 10 mM (pH 11), containing 10% methanol. Membranes were blocked overnight at 4 °C with PBS containing 0.1% Tween-20 (PBS-Tween) and 5% (w/v) low-fat milk powder. The membranes were incubated for 1 h at room temperature (RT) with the primary antibody in PBS containing 0.1% Tween-20 (PBS-Tween) and 1% (w/v) low-fat milk powder. The immune complexes were detected after incubation with the appropriate peroxidase-conjugated secondary antibody diluted (1:5000) in PBS-Tween, 2% low-fat milk, using the LumiSensor Chemiluminescent HRP substrate kit (Genscript; L00221V500), according to the manufacturer’s instructions. Protein expression of actin was used to correct the amount of each sample analyzed.
10
Protein Expression Detection Assay
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Protein electrophoresis was conducted on a Mini-PROTEAN system, and samples were transferred in a polyvinylidene difluoride membrane on a Mini Trans-blot cell (Biorad). Membranes were blocked for 1 hour in 5% non-fat dry milk in phosphate buffered saline (PBS)/Tween, followed by incubation with the appropriate primary antibody overnight. The next day membranes were washed 3×10 min in PBS/Tween and incubated with the appropriate secondary antibody for 1 hour. After three washes, proteins signals were visualised using the LumiSensor Chemiluminescent HRP Substrate Kit (Genscript, New Jersey, USA), in a UVP BioSpectrum Imaging System (UVP, California, USA). Data were analysed using ImageJ 1.51j8 software. Two-tailed t test was used to assess the p value.
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