Samples were prepared in M-Per Mammalian protein extraction reagent (ThermoScientific) according to manufacturer's instructions. For FACS-sorted somatic cells and oocytes, all cells obtained after each independent FACS experiment were processed separately. For embryonic gonads (13.5 dpc, 15.5 dpc, 18.5 dpc), for each independent experiment, 10–20 gonads were pooled to prepare the protein lysates. For postnatal ovaries, a pair of ovaries per animal was used to prepare each independent sample. Protein lysates were loaded onto 4%–15% SDS linear gradient gel (Bio-Rad). Standard blotting was performed using a nitrocellulose membrane with a semidry transfer machine at 16 V for 1 h (Bio-Rad). The membrane was blocked with 5% milk TBST solution for 30 min and then incubated with the primary antibody overnight at 4°C. The next day, three 10-min washes with TBST were performed. Incubation with secondary antibody (1:4000 anti-rabbit conjugated horseradish peroxidase; Life Technologies) diluted in 5% milk TBST solution was performed at room temperature for 1 h. Again, three 10-min washes with TBST were performed. Supersignal West Dura extended substrate (ThermoScientific) was used and chemiluminescent signals were detected using radiograph film. Antibodies used for immunoblot analyses are listed separately. Densitometry analysis was performed using ImageJ software where applicable.
Supersignal west dura extended substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Dura Extended Substrate is a chemiluminescent detection reagent for Western blotting. It is designed to produce a stable luminescent signal that can be detected using a CCD camera or X-ray film.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xr, by Bio-Rad (5 mentions)
Protran ba83, by Cytiva (3 mentions)
Pefabloc, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Nordic Biosite (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by GE Healthcare (2 mentions)
Anti ldlr antibody, by Progen Biotechnik (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
Anti her2, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti py1068 egfr, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked ab, by Cell Signaling Technology (1 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Chemidoc xrs apparatus, by Bio-Rad (1 mentions)
12 protocols using supersignal west dura extended substrate
1
Protein Extraction and Western Blotting
2
Immunoblotting Assay for EGFR and HER2
3
Quantifying Secreted and Intracellular PCSK9 Levels
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First, 48 h after transfection, supernatants of HEK293 cultured cells were collected and cells were lysed to determine secreted and intracellular PCSK9 levels by Western blot. Proteins from cell lysates or the supernatants were resolved by 8.5% Tris-Glycine SDS-PAGE, and gels were blotted onto nitrocellulose membranes (Protran BA 83, Whatman™, GE Healthcare, Munich, Germany), blocked for 1 h in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA, and immunoblotted with a mouse-anti-FLAG antibody (DYKDDDK tag rat monoclonal antibody, L5 clone; 1:1000 dilution) (Cat. No: MAI-142; Thermo Fisher Scientific, Invitrogen, Carlsbad, CA, USA) for 16 h at 4 °C. Detection was performed using a horseradish peroxidase-conjugated anti-rat antibody (Cat.No: 7077; Cell Signaling Technology® Inc., Danvers, MA, USA). Proteins were visualized using SuperSignal West Dura Extended Substrate (Thermo Fisher Scientific, Pierce, Carlsbad, CA, USA) on a ChemiDoc XRS apparatus (Bio-Rad, Hercules, CA, USA). Protein quantification was determined relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000 dilution) (Cat. No.: sc-26778, Santa Cruz Biotech. Inc., Dallas, TX, USA) using the NIH ImageJ software (https://rsbweb.nih.gob/ij/ (accessed on 12 January 2021)).
4
Quantification of Thrombin C-Terminal Fragments
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10 µl of citrate plasma was added to increasing concentration of DNA (6.25, 12.5, 25, and 50 µg/ml) resuspended in 10 mM Tris, pH 7.4 (total volume of 20 µl). Following an hour pre-incubation at 37 °C, 2.5 mM CaCl2 was added to samples and further incubated for 1 hour at 37 °C. After incubation, samples were boiled for 15 min with sample buffer and were analyzed by SDS-PAGE using Novex 10–20% Tris Tricine precast gels. Post electrophoresis, the gel was transferred to a PVDF membrane and blocked with 5% milk for an hour before western blotting. For detection of C-terminal thrombin fragments, overnight incubation of the blots was performed with polyclonal antibodies recognizing the thrombin C-terminal peptide VFR17 (VFRLKKWIQKVIDQFGE) in 1:5,000 dilution at 4 °C. Post washing thrice (3 x 10 mins) with PBS-T the blot was incubated with HRP-conjugated secondary antibodies (Dako) at 1:10,000 dilutions in 5% milk for an hour. After repeating the wash thrice, the membranes were developed using the SuperSignal West Dura Extended Substrate (ThermoFisher Scientific, USA) for 5 min and thereafter imaged using a gel documentation system (Gel Doc XR + System).
5
PCSK9 Expression and Secretion Analysis
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PCSK9 expression and secretion analysis in HEK293 cells transfected with empty plasmid and the different PCSK9 variants was performed by Western blotting. For that purpose, proteins from cell lysates or the supernatants were resolved by 8.5% Tris-Glycine SDS-PAGE. Gels were next blotted onto Nitrocellulose membranes (Protran BA 83, Whatman™, GE Healthcare, Munich, Germany), blocked for 1 h in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and immunoblotted with a rat monoclonal anti-FLAG antibody (1:1000) (Cat. No:MA1–142; Invitrogen, Thermo Fisher Scientific, Pierce, CA, USA) for 16 h at 4 °C. Then, they were counterstained with a horseradish peroxidase-conjugated goat anti-rat antibody (Cat. No: 7077S; Cell Signalling Technology® Inc., Danvers, MA, USA). The signal was created by adding SuperSignal West Dura Extended Substrate (Thermo Fisher Scientific, Pierce, CA, USA) and it was measured by the ChemiDoc XRS chemiluminescence system (Bio-Rad, Hercules, CA, USA) at 425 nm. The relativisation was performed using Glyceraldehide 3-phosphate dehydrogenase (GAPDH) as control.
6
Cell Lysis and Western Blot Analysis
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Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Roche Diagnostics, Mannhelm, Germany, 11873580001) and the 14,000Âg supernatants were used as cell lysates. Cell lysates and immunoprecipitants were resolved by SDS-PAGE and transferred onto PVDF membranes. The blots were incubated with primary antibodies followed by HRP-conjugated secondary antibodies. For detection, ECL prime Detection Kit (GE Healthcare, Little Chalfont, UK, RPN2232) or SuperSignal West Dura Extended Substrate (Thermo Scientific, Rockford, IL, USA, 34075) were used as chemiluminescent substrates and images were analyzed using LAS4000 mini (GE Healthcare).
7
Semi-Quantitative Immunoblotting of LDLR
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Cell lysates were prepared, protein concentration determined, and fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. Membranes were immunostained with rabbit polyclonal anti-LDLR antibody (1∶2000) (Cayman Chemical, Cat. No. 10007665) for 16 h at 4°C and rabbit polyclonal IgG anti-GAPDH antibody (1∶1000) (Santa Cruz Biotechnology, Cat. No. SC-25778) for 1 h at room temperature and counterstained with a horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signalling, Cat. No: 7074s). The signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA). ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) was used to detect the signals, and Quantity One Basic 4.4.0 software (Bio-Rad) was used to quantify band intensities. The concentrations of the antibodies were optimized to achieve low background and a linear dose-dependent increase in signal intensity. The relative band intensities for the mature and precursor forms of LDLR protein expressed for the different constructs was calculated as the ratio between the LDLR 160 kDa or 130 kDa bands to that of GAPDH.
8
Protein Extraction and Immunoblot Analysis
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Cells were lysed in ice cold 50 mM Tris-HCl buffer containing 125 mM NaCl, 1% Nonidet P-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/mL complete EDTA-free protease inhibitor cocktail (Roche), 0.25 mg/mL Pefabloc, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche), pH 7.5. Cells were rotated at 4 °C for an hour and centrifuged at 12,000 × g during 15 minutes to remove insoluble material. Protein fractionation was carried out by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative inmunobloting. Antibodies used were the following: rabbit polyclonal anti-LDLr antibody (1:500) (Progen Biotechnik GimbH, Heidelberg, Germany), anti-GAPDH antibody (1:1000) (Nordic Biosite, Little Chalfont, UK). Primary antibodies were incubated overnight at 4 °C, while secondary antibody was incubated at room temperature for 1 hour. Signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA) in a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). Band intensity quantification was performed using NHI ImageJ software (https://rsbweb.nih.gov/ij/ ) and levels of protein of interest were corrected to GAPDH.
9
Quantitative Immunoblotting of LDLr
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Cell lysates were prepared, using an ice-cold buffer containing 50 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Nonidet P-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/ml protease inhibitor cocktail (Roche), and 0.25 mg/ml Pefabloc, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche). Cells were sonicated for 10 pulses at 10 kHz on ice, rotated at 4°C for an hour and centrifuged at 12,000g during 15 minutes to remove insoluble material. Proteins were fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. The following antibodies were added: rabbit polyclonal anti-LDLr antibody (1:500) (Progen Biotechnik GmbH, Heidelberg, Germany), anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000) (Nordic Biosite, Täby, Sweden) and horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Little Chalfont, UK). The primary antibodies were incubated overnight at 4°C while the secondary antibody incubation was performed at room temperature for an hour. Signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA) in a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). NIH ImageJ software (http://rsbweb.nih.gov/ij/ ) was used for band intensity quantification, levels of protein were corrected to GAPDH loading control band intensities.
10
Protein Expression Quantification by Immunoblotting
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Cells were lysed using an ice‐cold buffer containing 50 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Nonidet P‐40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/ml complete EDTA-free protease-inhibitor cocktail (Roche), and 0.25 mg/ml Pefabloc, 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride (AEBSF; Roche). Cells were rotated at 4 °C for an hour and centrifuged at 12,000 g during 15 minutes to remove insoluble material. Proteins were fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. Following antibodies were added: rabbit polyclonal anti-LDLr antibody (1:500) (Progen Biotechnik GmbH, Heidelberg, Germany), anti-GAPDH antibody (1:1000) (Nordic Biosite, Täby, Sweden) and horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Little Chalfont, UK). The primary antibodies were incubated overnight at 4 °C while the secondary antibody incubation was performed at room temperature for an hour. Signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA) in a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). NIH ImageJ software (http://rsbweb.nih.gov/ij/ ) was used for band intensity quantification, levels of protein of interest were corrected to GAPDH loading control band intensities. Original blot can be found in Supplementary Fig. S1 .
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