Liver tissues were homogenized in lysis buffer (10 mM Tris-HCL, pH 8.0 and 1% SDS) containing phosphatase and protease inhibitor cocktails (P5726 and P8340, respectively; Sigma-Aldrich). These lysates were centrifuged at 15,000 rpm for 30 min at 20° C, and the protein concentration was determined using the Bradford method with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The lysates were subjected to SDS-PAGE electrophoresis (4–20% Criterion TGX Precast Gels; Bio-Rad Laboratories) and transferred to either a nitrocellulose or PVDF membrane (Trans-Blot Turbo Transfer pack; Bio-Rad Laboratories) using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). After blocking with 5% non-fat milk, the membranes were incubated overnight at 4° C with following antibodies (the catalog numbers are in parentheses). H-Ras (sc-520) and AMPKα 1/2 (sc-25,792) from Santa Cruz Biotechnology (Santa Cruz, CA) and ERK1/2 (9102), phospho-ERK1/2 (9101), AKT (9272), phospho-AKT (on Ser473; 9018), phospho-AMPKα (on Thr172; 2535) from Cell Signaling (Danvers, MA). All of the membranes were visualized using the Western Lightning ECL-Plus Kit (PerkinElmer, Waltham, MA). The band intensities were quantified using ImageJ software.
Western lightning plus ecl kit
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning Plus-ECL kit is a chemiluminescence-based detection system designed for the sensitive and quantitative detection of proteins in Western blotting applications. The kit provides reagents for the enhanced luminescent detection of target proteins, enabling researchers to obtain high-quality, low-background results.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (7 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Chemidoc touch imaging system, by Bio-Rad (5 mentions)
Tgx precast gel, by Bio-Rad (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Anti β actin hrp, by Merck Group (4 mentions)
Protease and phosphatase inhibitor, by CWBIO (3 mentions)
Sodium orthovanadate, by Merck Group (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Anti gapdh hrp, by Merck Group (3 mentions)
Anti erk2, by Santa Cruz Biotechnology (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Hrp conjugated anti rabbit igg, by Beyotime (3 mentions)
P8340, by Merck Group (2 mentions)
P5726, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Anti mouse igg antibody, by Southern Biotech (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
66 protocols using western lightning plus ecl kit
1
Western Blot Analysis of Liver Tissue Signaling
2
Skin Tissue Protein Extraction and Western Blot
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Skin tissue was homogenized in lysis buffer (10 mM Tris-HCl pH 7.5 and 1% SDS) containing phosphatase and protease inhibitor (P5726 and P8340; Sigma-Aldrich). Supernatants were collected after centrifugation and the protein concentration was determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Western blot analyses were performed as previously described26 (link). Briefly, the proteins were transferred onto nitrocellulose membranes and blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the antibodies described in Supplementary Table 2 . All membranes were visualized using the Western Lightning ECL Plus Kit (PerkinElmer, Waltham, MA, USA). The band intensities were quantified using NIH ImageJ software.
3
Western Blot Protein Quantification
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Total protein was extracted using RIPA lysis buffer (Servicebio, Wuhan, China) and quantified with a BCA protein assay kit (Solarbio, Beijing, China). After adding loading buffer, samples were boiled for 10 min, separated on SDS-PAGE, and transferred to PVDF (polyvinylidene fluoride) membranes. Membranes were blocked (5% skimmed milk powder in TBST for 2h at room temperature) and probed with primary antibodies overnight at 4℃. The antibodies used were against FGFR1 (Proteintech Cat# 60325-1-Ig, RRID : AB_2881435), E-CAD (Huabio, ET1702-53), N-CAD (Cell Signaling Technology Cat# 4061, RRID : AB_10694647), VIM (ABclonal Cat# A11883, RRID : AB_2772859), PARP (Cell Signaling Technology Cat# 9532, RRID : AB_659884), BAX (Cell Signaling Technology Cat# 5023, RRID : AB_10557411), BCL-2 (Cell Signaling Technology Cat# 2872, RRID : AB_10693462), and GAPDH (Proteintech Cat# 10494-1-AP, RRID : AB_2263076). Then, the membranes were incubated with IgG-horseradish peroxidase secondary antibodies (1:2000 dilution) for 2 h and finally washed three times, 10 min each time. The target proteins were detected using a Tanon 4800 imaging system (Yuanpinghao Biotechnology CO.LTD, Beijing, China) using the Western Lightning Plus ECL kit (PerkinElmer, Shanghai, China). Bands detected by Image Lab software.
4
Quantitative Western Blot Analysis of Hoxa10 and Hoxa11
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Western blotting was performed as described previously [13] (link). Twenty micrograms of total protein lysate was loaded in each lane. The primary antibody against mouse Hoxa10 (1:5000, GWB-CEOCD3) was obtained from GenWay (San Diego, CA, USA), whereas those for Hoxa11 (1:5000, NBP1-80228) and β-actin (1:10000, sc-47778) were obtained from Novus Biologicals (Littleton, CO, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The horse radish peroxidase (HRP)-conjugated secondary antibody (1:10000, C04001) was obtained from Croyez Bioscience (Taipei, Taiwan). Chemiluminescence was generated using a Western Lightning Plus ECL Kit (Perkin Elmer, Skovlunde, Denmark) and captured using X-ray film or Analytik Jena™ UVP ChemStudio PLUS and analyzed by VisionWorks Touch Software 9.0 (Analytik Jena US LLC, Upland, CA, USA).
5
Western Blot Analysis of Nfa34810 Signaling
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RAW264.7 cells were seeded into 6-well plates at a density of 1 × 106 cells/well and stimulated with Nfa34810 for various periods of time. The cells then were lysed with radioimmunoprecipitation assay buffer supplemented with phosphatase and protease inhibitors (CWBIO, China) on ice for 30 min. The supernatant was collected after centrifuging at 12,000 × g at 4°C for 25 min, and then the protein concentration was measured using a BCA protein assay kit (Tiangen Biotechnology, China). Equal protein concentrations were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies, including p-ERK1/2, p-p38, p-JNK, p-p65, p-AKT, NF-κB p-p65, and β-actin. Subsequently, membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Beyotime Biotechnology) or anti-mouse IgG antibodies (SouthernBiotech) and detected using a Western Lightning plus ECL kit (PerkinElmer, USA).
6
Immunoblot Analysis of Protein Samples
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Eluates were supplemented with RNase buffer to a final concentration 20 mM Tris–HCl pH 7.5, 150 mM NaCl and 200 U ml−1 of RNases A and T1. After 30 min incubation at 37 °C an aliquot of proteins was supplemented with LiDS loading buffer (Thermo Fischer Scientific) containing 20 mM DTT, separated on 4–12% polyacrylamide gels (NuPage, Thermo Fischer Scientific) and either stained with silver using Pierce Silver Stain Kit (Thermo Fischer Scientific) or electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes.
PVDF membranes were blocked in phosphate buffered saline supplemented with 0.1% Tween-20 (PBST) and containing 100 g L−1 milk powder. Membranes were then hybridized with antibodies diluted in PBST-milk that are listed inSupplementary Table 2 . Membranes were washed with PBST, hybridized with secondary antibodies conjugated with horseradish peroxidase (GE Healthcare) and developed using Western Lightning Plus ECL kit (PerkinElmer). Full-size images of the most important immunoblots are available in the Supplementary Fig. 1 .
PVDF membranes were blocked in phosphate buffered saline supplemented with 0.1% Tween-20 (PBST) and containing 100 g L−1 milk powder. Membranes were then hybridized with antibodies diluted in PBST-milk that are listed in
7
SDS-PAGE and Western Blot of AgRP
8
Protein Quantification and Western Blot Analysis
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Cell lysates were normalized to total protein content as determined by a DC protein assay (Bio-Rad). SDS-PAGE and Western blot analysis were performed using standard procedures with antibodies against NOX5 (Proteintech), Hsp90 (Abcam) and β-actin (Sigma-Aldrich). Horseradish peroxidase-linked secondary antibodies (anti-rabbit: Cell Signaling Technologies, anti-mouse: Abcam) were used to visualize the proteins using a Western Lightning Plus-ECL kit from PerkinElmer Life Sciences. Blots were imaged on a Bio-Rad ChemiDoc Imaging System and analyzed using Bio-Rad Image Lab software v5.2.1.
9
Protein Extraction and Western Blot Analysis
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Protein extraction from cells or tissue was performed using a lysis buffer that contained a protease inhibitor cocktail (Complete, Roche Diagnostics, cat. no. 04574834001). Total protein (25 μg) was loaded for each sample for separation by SDS–polyacrylamide gel electrophoresis (8%) and transferred onto nitrocellulose membranes (BioRad; cat. no. 1620115). Membranes were blocked with 5% non-fat milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBST) for one h and incubated in the presence of anti-Th (Abcam ab112 monoclonal anti-rabbit 1:1000), or anti-Th F-11 (sc-25269 monoclonal anti-mouse, 1:1000), or anti-Th Millipore ab152 (monoclonal anti-rabbit 1:1000), anti-Glial Fibrillary Acidic Protein (DAKO Z0334 polyclonal anti-rabbit 1:2000), anti-Beta-actin-peroxidase (Sigma A3854 monoclonal anti-mouse 1:25000). Proteins were revealed using secondary peroxidase-coupled anti-rabbit and anti-mouse antibodies (Jackson ImmunoResearch), using the Western Lightning Plus-ECL Kit (PerkinElmer; Waltham, MA). Images were digitally captured using the FUSION SOLO S instrument (Vilbert Smart imagining). Autoradiograms were analyzed by densitometry using the Image J® software (NIH).
10
Immunoprecipitation of IFT88 in Mitotic Cells
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LLC-PK1 and HCT116 cell extracts were obtained after lysis with buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% IGEPAL CA-630 and protease inhibitors (Sigma-Aldrich). Protein concentration for lysates was determined using Bradford reagent (Sigma-Aldrich), loads were adjusted and proteins were resolved by SDS-PAGE and analyzed by western-blot (Western Lightning Plus-ECL kit; PerkinElmer). For endogenous immunoprecipitations, LLC-PK1 cells were synchronized in mitosis using nocodazole and washout was done for 3 min at 37 °C. The resulting cell extracts were incubated for 2 h at 4 °C with IFT88 antibody or rabbit IgG (3 mg of total extracts and 2 µg of antibody) and then incubated for 45 min with protein G-PLUS agarose beads (Santa Cruz; sc-2002). Beads were washed three times with 500 μl lysis buffer and the immunoprecipitated proteins were separated by SDS-PAGE and analyzed by western blotting.
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