For protein analysis, CD14+ monocytes were seeded at a density of 1 × 106 cells/mL and cultured in the presence or absence of LPS (100 ng mL−1) for 24 h. Ice‐cold RIPA (Radio‐Immunoprecipitation Assay) buffer (Sigma‐Aldrich) containing 10 μg mL−1 phosphatase inhibitor cocktail and 10 μg mL−1 protease inhibitor cocktail (both Sigma‐Aldrich) was used to lyse cell suspensions. Total protein quantification and normalisation were performed using a BCA assay (Pierce Chemical Co., USA). Protein (1–2 μg) was resolved by SDS‐PAGE (5% stacking, 10% resolving), gels were then transferred onto PVDF membranes (Amersham Biosciences, UK) prior to 1 h blocking in wash buffer containing 3% BSA (0.1% Tween 20) with gentle agitation at room temperature. Membranes were incubated with mouse monoclonal anti‐HK2 (Novus Biologicals, USA), rabbit monoclonal anti‐PFKFB3 (Abcam, UK), rabbit polyclonal anti‐GLUT1 (Abcam), anti‐pSTAT3 and tSTAT3 (both Cell‐Signaling Technology, UK), and diluted in 3% BSA containing 0.1% Tween 20 at 4°C overnight with gentle agitation. β‐actin (1:5000, Sigma‐Aldrich) was used as a loading control. The signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Amersham Biosciences). Band densities were imaged using the ChemiDoc MP Imaging System (Bio‐Rad, USA). Densitometry analysis was carried out using ImageJ Software.
Supersignal west pico chemiluminescent substrate
Manufactured by Cytiva
Sourced in United States
SuperSignal® West Pico Chemiluminescent Substrate is a laboratory product designed for the detection of proteins in Western blot analyses. It is a chemiluminescent reagent that generates a luminescent signal when it reacts with the enzyme-labeled target proteins.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Mouse monoclonal anti hk2, by Novus Biologicals (2 mentions)
Ripa radio immunoprecipitation assay buffer, by Merck Group (2 mentions)
Edas 120 system, by Kodak (2 mentions)
T stat3, by Cell Signaling Technology (1 mentions)
Anti glut1, by Abcam (1 mentions)
Goat anti mouse secondary antibody, by LI COR (1 mentions)
Goat anti rabbit secondary antibody, by LI COR (1 mentions)
Imager 600 system, by Cytiva (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Apolyvinylidene fluoride membrane, by Merck Group (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Proteaseinhibitor cocktail 100x, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
6 protocols using supersignal west pico chemiluminescent substrate
1
Protein Analysis of LPS-Stimulated Monocytes
2
Ischemic Penumbra Protein Analysis
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After 24 h of reperfusion, rat brain tissue from the ischemic penumbra was collected and lysed in high-efficiency RIPA lysis buffer containing PMSF. The protein concentration was determined using a BCA assay. Equal amounts of protein were loaded and separated on 10–12% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes in Tris-glycine transfer buffer. The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with a primary antibody against β-actin (1:1000), β-tubulin (1:1000), Iba1 (1:1000), CD40 (1:1000), CD68 (1:1000), CD206 (1:1000), GPER (1:1000), P65-NF-κB (1:1000), p-P65-NF-κB (1:1000), ERK (1:1000), or p-ERK (1:1000) at 4 °C overnight. The membranes were then incubated with a goat anti-mouse secondary antibody (1:5000; LI-COR, USA) or goat anti-rabbit secondary antibody (1:5000; LI-COR, USA) at room temperature for 1 h. The bands corresponding to the antigen–antibody complexes were detected using Super Signal West Pico Chemiluminescent Substrate and visualized with an Amersham Imager 600 system.
3
Western Blot Protein Analysis Protocol
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Cells lysate was prepared using T-PER Tissue Protein Extraction Reagent (Thermo
Scientific) supplemented with Halt™ phosphatase and protease
inhibitor cocktail (100X) (Thermo Scientific). Proteins were separated on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim
milk, the membrane was incubated with primary antibodies, followed by incubation
with horseradish peroxidase-conjugated secondary antibody. Target proteins were
visualized using Super Signal West Pico Chemiluminescent Substrate and
ImageQuant 800 (Amersham).
Scientific) supplemented with Halt™ phosphatase and protease
inhibitor cocktail (100X) (Thermo Scientific). Proteins were separated on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim
milk, the membrane was incubated with primary antibodies, followed by incubation
with horseradish peroxidase-conjugated secondary antibody. Target proteins were
visualized using Super Signal West Pico Chemiluminescent Substrate and
ImageQuant 800 (Amersham).
4
Protein Extraction and Western Blot Analysis
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Cells were scraped and centrifuged prior to cell lysis. Ice-cold RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma, Missouri, USA) containing 10 μg/ml phosphatase inhibitor cocktail and 10 μg/ml protease inhibitor cocktail (Sigma) was used to extract protein from transfected HUVEC. Measurement of protein concentration was performed using a BCA assay (Pierce Chemical Co, Rockford, IL, USA). Protein (5 μg) was resolved on SDS-PAGE (5% stacking, 10% resolving), gels were then transferred onto PVDF membranes (Amersham Biosciences, UK) prior to 1 h blocking in wash buffer containing 3% BSA (0.1% Tween) with gentle agitation at room temperature. Membranes were incubated with mouse monoclonal anti-HK2 (Novus Biologicals, Colorado, USA) and rabbit monoclonal anti-PFKFB3 (Abcam, UK), and diluted in 3% BSA containing 0.1% Tween 20 at 4 °C overnight with gentle agitation. β-actin (1:5000, Sigma) was used as a loading control. Following three 15 min washes, membranes were incubated with appropriate horseradish peroxidase conjugated secondary antibodies (1:5000) for 3 h at RT. The signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Amersham Biosciences). Band densities were imaged using the ChemiDoc MP Imaging System (Bio-Rad, California, USA).
5
Profiling Synovial Fibroblast Signaling in Psoriatic Arthritis
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Protein isolation from Psoriatic Arthritis synovial fibroblasts (PsAFLS) and synovial explants is described in online supplementary file 1. Protein (20–50 µg) was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking, 10% resolving), gels were then transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK) prior to 1 h blocking in wash buffer containing 5% non-fat milk. Membranes were incubated with rabbit polyclonal anti-pSTAT3 (Cell-Signaling Technology, UK), total-signal transducer and activator of transcription (tSTAT)3, pSTAT1, tSTAT1, pSTAT2, suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3; Cell Signaling Technology) and nuclear factor kappa B cells (NFκBp65) (Millipore, California, USA) diluted in 5% non-fat milk containing 0.1% Tween 20 at 4°C overnight. β-Actin (Sigma-Aldrich) was used as a loading control. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 3 h at RT. Signal was detected using SuperSignal West-Pico Chemiluminescent Substrate (Amersham Biosciences, UK) and quantified using EDAS-120 system (Kodak, Rochester, New York, USA).
6
Western Blot Analysis of Protein Expression
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Protein expression was evaluated by western blot, using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) techniques as previously described (8 (link)). Briefly, harvested cells were lysed in 5X Laemmli sample buffer (5 mmol/L Na3VO4, 2.5 mmol/L p NPP, 10 mmol/L NaF, 5 mmol/L EDTA, 5 mmol/L EGTA, 20 μg/L leupeptin, 20 μg/L pepstatin and 20 μg/L aprotinin) and incubated for 3 min at 70°C and stored at −80°C until use. Lysates were sonicated and subjected to 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose and blocked in 5% milk in Tris-buffered saline containing Tween 20 (TBS-T) for 1 h. The membranes were then probed overnight with primary antibodies-anti-gp91/NOX2 (Santa Cruz 1:5,000), anti-NOX5 (Abcam 1:1,000), and anti-actin (Sigma 1:10,000). Membranes were washed and incubated with HRP-labeled specific antibody. The signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Amersham Biosciences) and density of the bands was analyzed using EDAS 120 system from Kodak (Kodak, Rochester, NY, USA).
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