Treated cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in a minimum volume of 1X cell lysis buffer [1% Nonidet P-40; 50 mM Tris pH 7.4; 10 mM NaCl; 1 mM NaF; 5 mM MgCl2; 0.1 mM EDTA; 1 mM PMSF; and COMPLETE™ protease inhibitor cocktail tablet (Roche Diagnostocs Corp.)]. Protein content was determined using the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Cell extracts (10 μg/lane) were resolved by 4–12% gradient SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and electro-transferred onto nitrocellulose membranes. Following the transfer, membranes were stained with Ponceau S to confirm equal protein loading. Membranes were blocked with PBS/0.1% Tween-20 (PBST) and 10% skim milk and incubated with antibodies in PBST overnight at 4°C. Following incubation with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies, signals were detected using the SuperSignal Chemiluminescent Reagent (Pierce Chemical Co., Rockford, IL) with exposure of blots onto X-ray films.
Supersignal chemiluminescent reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal Chemiluminescent reagents are a series of products designed to detect and quantify proteins and other biomolecules in Western blot and related immunoassays. The reagents generate a light signal in the presence of the target analyte, which can be measured using a luminometer or imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Gradient sds polyacrylamide gel electrophoresis, by Bio-Rad (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase linked hrp rabbit secondary antibody, by GE Healthcare (2 mentions)
β actin, by Merck Group (2 mentions)
Rho gtpase antibody sampler kit, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Phospho stat antibody sampler kit, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Phospho mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions)
Ab218528, by Abcam (1 mentions)
Sc 69778, by Santa Cruz Biotechnology (1 mentions)
Sc 271064, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
C met, by Merck Group (1 mentions)
Myogenin, by Novus Biologicals (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Total stat3, by Cell Signaling Technology (1 mentions)
15 protocols using supersignal chemiluminescent reagent
1
Western Blotting for Protein Analysis
2
Western Blotting for Protein Analysis
3
Protein Expression and Signaling Analysis
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Whole proteins from the cultured cells were extracted according to the manufacturer’s protocol (RIPA) (Biyuntian, Shanghai, China), followed by concentration investigation. All samples were separated by SDS-PAGE and transferred following standard protocols. Antibodies to CD117 (Abcam, Cambridge, MA, USA) and FcεRI (Abcam) were used to identify mature mast cells. Rho-GTPase Antibody Sampler Kit (Cell Signaling Technology, Danvers, MA, USA), Phospho-MAPK Family Antibody Sampler Kit (Cell Signaling Technology) and Phospho-Stat Antibody Sampler Kit (Cell Signaling Technology), were used to detect the activation of signaling pathways in colon tumor cells. Antibody to cleaved caspase3 (Millipore, Temecula, CA, USA) was used to identify cell apoptosis. Antibody to β-tubulin (Cell Signaling Technology) was used to ensure the consistency of each cell lysate. Antibody to Pseudomonas Exotoxin A (Sigma-Aldrich Corp., St. Louis, MO, USA) and mouse anti-Human IgE antibody (Abcam) were used to identify the recombinant protein toxin. HRP-conjugated secondary antibodies were purchased from Cell Signaling and SuperSignal chemiluminescent reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Protein expression was evaluated using Quantity One software according to immunoblotting band intensity.
Protein expression was evaluated using Quantity One software according to immunoblotting band intensity.
4
Western Blot Analysis of ABCG1, IRAK-1, RXR, and LXR
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Cells were harvested and lysed in RIPA buffer. Protein content was measured using a BCA assay (Beyotime Institute of Biotechnology). Protein (50 µg) was separated via 10% SDS-PAGE and transferred to poly-vinylidene difluoride membranes, which were washed in Tris-buffered saline with Tween-20 (TBST) for three times and blocked with 5% non-fat in TBST for 1 h at room temperature. Subsequently, the membranes were incubated with antibodies against ABCG1 (ab218528, 1:1,000, Abcam), IRAK-1 (sc-515512, 1:1,000, Santa Cruz Biotechnology, Inc.), retinoid X receptor (RXR, sc-46659, 1:1,000, Santa Cruz Biotechnology, Inc.), liver X receptor (LXR, sc-271064, 1:1,000, Santa Cruz Biotechnology, Inc.) and GAPDH (sc-69778, 1:5,000, Santa Cruz Biotechnology, Inc.) at 4°C overnight. The membrane was washed three times with TBST for 15 min and incubated with a horseradish peroxidase-conjugated secondary antibody (sc-2004, 1:5,000, Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. Proteins were detected by Super Signal chemiluminescent reagent (Thermo Fisher Scientific, Inc.) and measured using Flowjo 7.6.1 (FlowJo, LLC).
5
Immunoblotting analysis of JKAP signaling
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Anti-JKAP antibody (clone #3D3) purchased from Abnova, anti-phospho-Y394-Lck antibody (#2101) purchased from Cell Signaling, anti-myc antibody (#9E10) purchased from Millipore, and anti-β-actin antibody (#AC-74) purchased from Sigma-Aldrich were used for immunoblotting. GFP-tagged JKAP (WT) and JKAP C88S plasmids were reported previously [14 (link)]. The SuperSignal chemiluminescent reagent was from Thermo. The rest of the chemicals were purchased from Sigma-Aldrich.
6
Analyzing Muscle Injury Protein Changes
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About 100 μg of protein derived from crushed muscle extracts and uninjured muscle were run out on a separate acrylamide gel, transferred to PVDF membrane (BioRad, Mississauga, Ontario), blocked with 5% skim milk for 1 h at RT, and then incubated overnight at 4°C with primary Active-HGF antibody (Abcam). A similar western blot protocol was completed for the analysis of c-met (Sigma Aldrich) and myogenin (Novus Biologicals) protein content, using 30 μg of protein from lysates of uninjured and 5 days post-injury muscles, respectively. The appropriate horseradish peroxidase-conjugated secondary antibodies were incubated for 1 h at RT, and the blot was visualized using SuperSignal Chemiluminescent reagent (Thermo Scientific). Images were acquired using a Gel Logic 6000 Pro Imager (Carestream, Rochester, NY) and the area density of each band was analyzed using Adobe Photoshop.
7
STAT3 Activation Immunoblotting
8
Western Blot Analysis of FLAG-Tagged Proteins
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Cells were pelleted, washed once in 1X PBS, and lysed by bead-beating. 6X Laemmli buffer was added to the whole cell lysate and then boiled for 10 minutes. Proteins were separated on a 12–14% Tris-Glycine SDS PAGE gels (BioRad, Hercules, CA), transferred to PVDF membrane (Pall Corp, Pensacola, FL), incubated with anti-FLAG antibodies (Sigma Aldrich, St. Louis, MO) at 1:15,000 dilution, washed thrice in 1X PBST, and incubated with a 1:1000 dilution of a secondary antibody conjugated to HRP (Pierce, Rockford, IL). Membranes were incubated with SuperSignal chemiluminescent reagent (Thermo Fisher Scientific, Rockford, IL) for 5 minutes in the dark and imaged on a FluorChem8900 (AlphaInnotech, Santa Clara, CA).
9
Immunoprecipitation and Western Blotting
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The immunoprecipitates and total cell lysates were subjected to electrophoresis on 8% or 10% acrylamide gels. After electrophoresis of immunoprecipitates, gels were dried and analyzed by electronic autoradiography. For western blotting, proteins in the gels were transferred to nitrocellulose filters (Amersham, Piscataway, NJ) and probed with the primary antibodies specified in the figure legends after blocking with 5% dried milk and HRP-conjugated anti-rabbit or anti-mouse IgG (Amersham). The antigen-antibody complexes were visualized by SuperSignal Chemiluminescent reagents (Thermo Scientific). The specific intensity of each protein band on X-ray film was measured by IMAGE J software from the National Institutes of Health and expressed as a ratio of the optical density band of each protein to that of β-actin.
10
Hypoxia Response Protein Analysis
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Worms were synchronized by hypochlorite extraction of eggs and exposed to control or experimental conditions beginning on day 2 of adulthood. Protein was extracted 24 h later by 3 rounds of freeze-thaw cracking and dounce homogenization in a homogenization buffer [15 mM Hepes (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA 44 mM, 1 mM DTT, 2 mM NaVO4, 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, 1x Phosphatase Inhibitor Cocktail 2 (Sigma)]. Samples were run on a 4–12% Bis-Tris gel in MOPS buffer and transferred to a PVDF membrane. Blots were blocked for 15 min in 5% nonfat dehydrated milk and probed for 1 h with either a mouse α-HIF-1 antibody in whole serum provided by Dana Miller (Budde and Roth, 2010 (link)) or mouse α-actin antibody MAB150 (Millipore) diluted 1:10000 in 1% milk. Santa Cruz Biotechnology secondary HRP-conjugated goat α-mouse IGG antibody (sc-2064) was diluted 1:5000 in 1% milk and incubated for 1 h. SuperSignal chemiluminescent reagents (Thermo Scientific) were used as HRP substrate according to the manufacturer's recommended procedure. Abcam ab69312 and Santa Cruz Biotechnology sc-9996 were used at a 1:2500 dilution for anti-GFP immunoblotting.
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