Radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) was used to lyse samples, which were then spun at 12,000 rpm for 15 min at 4°C. Supernatant protein levels were then assessed via bicinchoninic acid (BCA) assay (Beyotime), and 40 ng protein from each sample was separated via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA) that were blocked for 2 h using 5% nonfat milk, after which they were probed overnight using primary antibodies specific for aggrecan, collagen II, β-actin, Nrf2 (1:500), HO-1 (1:5,000), COX-2, iNOS, MMP-13, ADAMTS-5, p65, IκBα, and Lamin B1 at 4°C. Antibodies were used at a 1:1,000 dilution unless otherwise indicated. After washing, blots were then probed for 2 h with appropriate secondary antibodies were visualized via enhanced chemiluminescence (Invitrogen), with Image Lab 3.0 (Bio-Rad) used to quantify band density.
Enhanced chemiluminescence
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Germany, Japan, Sweden, Israel
Enhanced chemiluminescence is a laboratory technique used to detect and quantify proteins in biological samples. It involves the use of chemiluminescent substrates that emit light when they react with specific enzymes, allowing for the visualization and measurement of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (89 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (44 mentions)
Polyvinylidene fluoride membrane, by Merck Group (37 mentions)
Protease inhibitor cocktail, by Merck Group (33 mentions)
Polyvinylidene difluoride membrane, by Merck Group (27 mentions)
Bca protein assay kit, by Beyotime (26 mentions)
Ripa lysis buffer, by Beyotime (25 mentions)
Ripa buffer, by Thermo Fisher Scientific (24 mentions)
Ripa buffer, by Beyotime (21 mentions)
β actin, by Santa Cruz Biotechnology (21 mentions)
β actin, by Cell Signaling Technology (20 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (20 mentions)
Bca protein assay, by Thermo Fisher Scientific (20 mentions)
Protease inhibitor cocktail, by Roche (19 mentions)
Nitrocellulose membrane, by Bio-Rad (19 mentions)
Pvdf membrane, by Bio-Rad (18 mentions)
β actin, by Merck Group (18 mentions)
Gapdh, by Cell Signaling Technology (17 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (17 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (16 mentions)
875 protocols using enhanced chemiluminescence
1
Western Blot Analysis of Chondrocyte Proteins
2
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed in ice-cold RIPA lysis buffer containing protease inhibitor (Beyotime, China) and incubated on ice for 30 min. Then, cell lysates were centrifuged, and the supernatants were transferred to a new tube for protein concentration determination (BCA assay, Beyotime Biotechnology, China). A 5× loading buffer was added to the sample to obtain a 1× final solution. The samples were then heated at 95°C–100°C for 10 min and cooled to room temperature before separation by SDS-PAGE. Proteins were separated on gels, transferred to PVDF membranes (Merck Millipore), and blocked in 5% skim milk at room temperature. The membranes were first incubated in primary antibodies at 4°C overnight and then in secondary antibodies at room temperature for 1 h. The signal was visualized with enhanced chemiluminescence (Invitrogen, Carlsbad, CA, United States). The relative expression was quantified using Image Lab 6.0 and normalized to blots of negative control (NC). The antibodies used were as follows: eIF3a (Abcam, ab128996); Gapdh (Sigma, SAB1405848); Cdc42 (Proteintech, 10155-1-AP); Rhoa (Proteintech, 10749-1-AP); ROCK1 (WL01761); ROCK2 (WL00500); and cortactin (ab81208).
3
Apoptosis Pathway Activation in Cancer Cells
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H460, H1975 and HCC827 cells were exposed to DMSO, HCPT (0.125 or 0.5 μM), CRI (1 or 2 μM) or the combination for 48 h or 72 h. The proteins were collected after cell lysis and centrifugation. The protein concentration was determined by Coomassie blue method. The proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. After blocked with 5% skimmed milk, the membranes were immunoblotted with the primary antibodies (dilution of 1:1000) against Bax, Bcl-2, Bcl-xL, Cyto-C, Casp-8, Cleaved-Casp-8, EGFR, p-EGFR, AKT, p-AKT, ERK, p-ERK, JNK, p-JNK, p38 MAPK, p-p38 MAPK and α-Tubulin overnight at 4 °C. Then secondary antibodies-conjugated with horseradish peroxidase (HRP) were applied (dilution of 1:2000) in blocking buffer for 1 h. HRP -conjugated goat anti-rabbit or anti-mouse IgG was used as a secondary antibody for enhanced chemiluminescence (Invitrogen). The blots were visualised by Odyssey infra-red imaging system (LI-COR Biotechnology, USA).
4
Protein Detection in Cell Lysates
5
Thylakoid Membrane Protein Isolation and Analysis
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Thylakoid membrane proteins were isolated from 4-week-old WT seedlings as described previously (Zhang et al. 1999 (link)). BN-PAGE and two-dimensional analysis were performed as described by Peng et al. (2006 (link)), and immunoblot analysis according to our previous study (Chen et al. 2016 (link)). Thylakoid proteins were transferred to PVDF membranes (Millipore) after electrophoresis and probed with relevant antibodies specific to PSI subunits PsaA, PsbA (D1), and CP47 (PsbB), cpATPase subunits AtpA and AtpB, and LHCII. Signals were visualized by enhanced chemiluminescence (Invitrogen). Samples were loaded based on an equal chlorophyll content in both BN-PAGE and immunoblot analysis.
6
Nicotinic Acetylcholine Receptor Expression Analysis
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The heart of mice from each group was used for western blotting. Briefly, they were homogenized using a radioimmunoprecipitation lysis buffer (Sigma, St Louis, USA) containing a proteinase inhibitor (Solarbio, Beijing, China) and centrifuged for 10 min at 4°C. The supernatant was further centrifuged for 20 min. A protein assay kit (Thermo Fisher, Waltham, USA) was then used to assess the protein concentration of each supernatant. Samples with equal amounts of protein were separated using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad). They were then transferred onto an Immobilon membrane (Millipore, Billerica, MA), and after blocking with BlockAce (Dainippon Pharmaceutical, Japan), primary antibodies, including polyclonal antibody for α7 nicotinic acetylcholine receptor (α7nAchR, Abcam, Cambridge, England), and β-actin (internal control, Cell signal Technology) were used at the recommended concentration based on the manufacturer's instructions and dilution ratio of 1 : 3000. The proteins were then detected using enhanced chemiluminescence (Invitrogen). The bands were analyzed on the ECL detection system (GE Healthcare). All experiments were performed in triplicate and repeated at least three times. The quantitative analysis was the ratio of the target protein and β-actin for western blot.
7
Western Blot Analysis of Protein Expression
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Cells were harvested by trypsinization, lysed in 1× sodium dodecyl sulfate lysis buffer, and denatured for 10 min at 100 °C. After immunoblotting, the membranes were blocked with 5% nonfat dry milk in TBS/0.1% Tween-20 and then incubated with the primary antibodies in 1% nonfat dry milk in TBS/0.1% Tween-20. Subsequently, the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) and visualized with enhanced chemiluminescence (Invitrogen, Carlsbad, CA).
The immunoreagents used for Western blotting were rabbit polyclonal antibody against Gli1 (Abcam, ab92611, diluted 1:500) and rabbit polyclonal anti-FoxM1 (Abcam, ab137647, diluted 1:500). Mouse monoclonal antibody against CCNB1 was purchased from Cell Signaling Technology (CST, 4135, diluted 1:2000), and anti-β-actin antibody (Anbo, E0012, diluted 1:5000) or anti-GAPDH antibody (Millipore, MAB374, diluted 1:2000) was used as a loading control.
The immunoreagents used for Western blotting were rabbit polyclonal antibody against Gli1 (Abcam, ab92611, diluted 1:500) and rabbit polyclonal anti-FoxM1 (Abcam, ab137647, diluted 1:500). Mouse monoclonal antibody against CCNB1 was purchased from Cell Signaling Technology (CST, 4135, diluted 1:2000), and anti-β-actin antibody (Anbo, E0012, diluted 1:5000) or anti-GAPDH antibody (Millipore, MAB374, diluted 1:2000) was used as a loading control.
8
Western Blot Analysis of Cell Signaling Proteins
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Cells or tissues were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Following centrifugation at 12,000 × g for 15 min, total proteins were separated using 10% SDS-PAGE (Pierce Chemical Company, Rockford, IL, USA) and transferred to PVDF membranes (Millipore, Boston, MA, USA). The membranes were blocked for 1 h in PBS and 5% nonfat milk, followed by overnight incubation at 4°C with the primary antibodies against ALX4, E-cadherin, fibronectin, N-cadherin, Shh, Ptch1, Gli1, or β-actin. After washing three times with TBST, the membranes were incubated for 1 h at 37°C with secondary HRP-conjugated antibodies. All antibodies used in these experiments were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunoreactive protein bands were visualized with enhanced chemiluminescence (Invitrogen). The relative protein expression levels were analyzed using Glyko BandScan 5.1 software (Life Technologies, Gaithersburg, MD, USA). β-Actin was chosen as an internal control.
9
Pancreatic Protein Extraction and Western Blot
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Mouse pancreatic tissue (100 mg) was homogenized in 0.5 ml lysate buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 100 mM Na4P2O7, 2 mM Na3VO4, 100 mM NaF and 1% Nonidet P-40 and protease inhibitors were added at a fixed volume ratio [lysate buffer:phenylmethylsulfonyl fluoride (PMSF):protease inhibitor cocktail, 100:1:1]. Homogenized samples were centrifuged at 4°C, at 14,000 rpm for 15 min. The concentrations of protein from the supernatant were determined using BCA protein assay reagent. Protein (25 μg) was loaded for 10% sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). After blocking with 5% bovine serum albumin, immunoblotting was carried out with various primary antibodies at 4°C overnight or at 25°C for 2 h (anti-GAPDH antibody was used as an internal control). After incubation with IgG horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, the membranes were washed three times successively. Then the specific proteins were detected by enhanced chemiluminescence (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The relative band intensity was quantified by Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
10
Western Blot Protein Analysis
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Cells were lysed using RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). The debris were removed from the lysates by centrifugation and protein estimation was performed by Bicinchoninic Acid (BCA) assay (Sigma). Proteins were resolved by loading 10ug of protein on a 10% and 12% SDS-PAGE gel. Transfer of proteins to the polyvinylidene difluoride (PVDF) membrane was done using a wet transfer technique. Blots were probed with primary antibodies, indicated in Table 1 , for overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 1 hr at room temperature, indicated in Table 1 . The protein bands were visualized with enhanced chemiluminescence (Invitrogen/Thermo scientific). The uncropped western blots are shown as supplemental data.
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