Cells were harvested and lysed by cell lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktails) on ice for more than 15 minutes. Cell lysate was centrifuged for 10 minutes at 13,000 rpm at 4°C, and the supernatant was transferred to a new tube for BCA protein quantification assay. Equal amounts of protein sample were added into 4× sample buffer and boiled for 5 minutes. The sample was subjected to SDS-PAGE analysis and transferred to nitrocellulose membrane. The membrane was blocked by 5% milk for 1 hour at room temperature and incubated with primary antibody at 4°C overnight. The membrane was washed six times with 1× TBST and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. The protein was visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific).
Supersignal west pico stable peroxide solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
SuperSignal West Pico Stable Peroxide Solution is a laboratory reagent used for the detection of proteins in Western blot analysis. It is part of the SuperSignal West Pico Chemiluminescent Substrate kit and is designed to provide a stable and consistent signal for the detection of low-abundance proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
T7 polymerase, by New England Biolabs (3 mentions)
Biotin rna labeling mix, by Roche (3 mentions)
Supersignal west pico luminol enhancer solution, by Thermo Fisher Scientific (3 mentions)
Sample buffer, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Bca protein quantification assay, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd63 primary antibody, by BD (1 mentions)
Mouse anti human cd81, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Luminol enhancer solution, by Thermo Fisher Scientific (1 mentions)
Criterion 10 tris hcl gel, by Bio-Rad (1 mentions)
30 protocols using supersignal west pico stable peroxide solution
1
Western Blot Protein Quantification
2
Western Blot Analysis of Exosomal Markers
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Samples were lysed with RIPA buffer (Thermo Fisher, #89900). Protein concentration was determined by bicinchonic acid assay. 100 µg of protein from each sample was loaded onto a Criterion 10% Tris-HCl gel (Bio-Rad, Hercules, CA, cat #3451018) and electrophoresed. The proteins were then transferred to a PVDF membrane (Bio-Rad, cat #1620174) and blocked with 5% powdered milk (Bio-Rad, cat #1706404) in Dulbecco’s PBS (Thermo Fisher, cat #14190250) +0.1%Tween®20 (Sigma-Aldrich, St. Louis, MO, cat #P9416) for 1 h. The membrane was subsequently incubated with mouse-anti-human CD63 primary antibody (BD, cat #556019), at a 1:1000 dilution for 1 hour, and mouse-anti-human CD81 (Santa Cruz Biotechnology, cat #sc-166029), at a 1:1000 dilution for 1 hour. After washing the membrane, it was incubated with a goat-anti-mouse IgG-HRP secondary antibody (Santa Cruz Technology, cat #sc-2005) at a 1:10,000 dilution for 1 h. The membrane was then incubated with a 1:1 mixture of SuperSignal West Pico Stable Peroxide solution (Thermo Fisher, cat #34080) and Luminol Enhancer solution (Thermo Fisher, cat #34080) for 5 min. The membrane was visualized on Amersham HyperfilmTM ECL chemiluminescence film (GE Life Sciences, PA, cat #28906839).
3
Western Blot Protein Analysis
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Cells were harvested and lysed in cell lysis buffer on ice for more than 15 minutes and the lysate was centrifuged at 13,000 rpm at 4 ℃ for 10 minutes. The supernatant was transferred into a new tube for BCA protein quantification assay (Thermo Fisher Scientific). 4x loading buffer (Thermo Fisher Scientific) was added into equal amount of protein sample and boiled for 5 minutes. The sample was used for SDS-PAGE analysis and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in room temperature for 1 hour and incubated with primary antibody at 4 ℃ overnight. The membrane was washed with 1x TBST 5 minutes for 3 times, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour in room temperature. The protein was visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific).
4
Immunoprecipitation and Western Blot Analysis
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For IP, cells were harvested and lysed in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 1% protease inhibitor cocktails, Sigma-Aldrich) using glass dounce homogenizer. Cell lysates were centrifuged and the supernatant was incubated with indicated antibodies and protein-G beads (Invitrogen) at 4°C overnight. The beads were washed five times with cell lysis buffer and the precipitated proteins were subjected to further analysis. For western blotting, protein samples were prepared in modified RIPA buffer (1×PBS, 1% NP-40, 0.1% SDS and 1% protease inhibitor cocktail). Equal amounts of protein (50 ~ 100 μg) from cell lysates were denatured in sample buffer (Invitrogen), subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by Super Signal West Pico Stable Peroxide Solution (Thermo Scientific).
5
Western Blot Protein Analysis Protocol
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Cells (1×106) were lysed with lysis buffer (1% Nonidet P-40, 1X PBS, 0.1% sodium dodecyl sulfate and 1% protease inhibitor cocktail), followed by protein quantification using a bicinchoninic acid (BCA) assay. Samples were diluted in loading buffer containing dithiothreitol and boiled for 5 min. Equal amounts (50 µg) of protein for each sample was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were immuno-blotted with the aforementioned specific primary antibodies targeted at the protein of interest in 4°C overnight. Subsequently, the membrane was wash three times with 1X TBST and incubated with rabbit IgG (cat. no. MR-R100; 1:3,000) and (mouse IgG; cat. no. MR-M100; 1:3,000) horseradish peroxidase-conjugated secondary antibodies (both Shanghai MRbiotech, Co., Ltd., Shanghai, China) for 1 h at room temperature, and then visualized using SuperSignal West Pico Stable Peroxide solution (Thermo Fisher Scientific, Inc.).
6
RNA-Protein Interaction Identification
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RNAs were biotin-labeled during in vitro transcription using Biotin RNA Labeling Mix (Roche) and T7 polymerase (New England Biolabs). P-18 primary cells cultured in androgen-depleted medium were lysed in modified Binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 1% protease inhibitor cocktails). Cell lysates were incubated with biotin-labelled RNAs and streptavidin beads at 4 °C for 12 h. The beads were washed in wash buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 0.05% Nonidet P-40 (NP-40); 1 mM MgCl2) at 4 °C six times. The samples were subjected to western blot analyses as described previously [34 (link)]. Briefly, protein samples were denatured and subjected to SDS-polyacrylamide gel electrophoresis (SDS/PAGE), and were transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Scientific). The antibodies are shown in Table S1 .
7
Western Blot Protein Analysis
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The collected cells were lysed in Laemmli sample buffer (Bio-Rad). Whole cell lysates were boiled for 10 minutes and then centrifuged for 10 minutes to remove cellular debris. The protein concentrations were quantified with a Pierce BCA Protein Assay kit (Thermo Scientific; Rockford, IL). Fifty μg of total protein were separated on 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyl difluoride (PVDF) membranes. The membranes were blocked at room temperature for 1 hour in 5% nonfat dry milk, incubated with the indicated primary antibodies overnight at 4°C, washed in TBST buffer, and then incubated for 1 hour with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. The membranes were washed in TBST buffer again and immune complexes were finally detected with SuperSignal West Pico stable peroxide solution (Thermo Scientific). The protein bands were visualized using FluorChemE imager.
8
Immunoprecipitation and Western Blotting Protocol
9
Western Blot Analysis of Prostate Cancer Cells
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Prostate cancer cells were harvested and lysed by RIPA buffer on ice, the supernatant was quantified by BCA protein quantification assay (Bio-Rad, USA). Equal amounts of protein sample were added to 4× sample buffer and boiled for 10 min. The sample was subjected to SDS-PAGE analysis and transferred to nitrocellulose membrane. The membrane was blocked by 5% milk for 2 h at room temperature and incubated with primary antibody at 4°C overnight. The second day, the membrane was washed three times with 1 × TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized by super signal West Pico Stable Peroxide Solution (Thermo Fisher Scientific, USA).
10
Western Blot Protein Analysis
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Protein samples were prepared by lysing cells in RIPA buffer supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). The concentration of samples was measured by BCA assay kit (Thermo Fisher Scientific). Equal amounts of protein (50-100 μg) from cell lysates were denatured at 95 oC for 10 mins, prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies (diluted at 1:1,000 to 1:5,000) at 4 oC overnight, followed by horseradish peroxidase (HRP) conjugated secondary antibody (diluted at 1:5,000) incubation at room temperature for 1 h. The protein signals were visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific).
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