T24, 5637 and EJ cells were harvested and lysed in lysis buffer (150 mM KCl, 75 mM HEPES (pH 7.5), 1.5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 0.075% NP-40 supplemented with protease inhibitor cocktail [Roche, USA]). The lysate was precleared using a mixture of protein A–Sepharose (CL-4B; GE Healthcare) and the appropriate antibody overnight at 4 °C. Immunoprecipitates were washed with lysis buffer, resuspended in sample buffer, boiled and analyzed by SDS–PAGE. Individual samples (40 μg of protein) were separated on an 8% SDS polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in a PBS-Tween 20 solution with 5% fat-free milk for 1 h at room temperature and were then incubated with appropriate dilutions of primary antibodies specific for STAT3 overnight at 4 °C. After washing, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG for 1 h. Membranes were developed in an ECL mixture (Vector Laboratories, Burlingame, CA) and visualized by Imager.
Ecl mixture
Manufactured by Vector Laboratories
The ECL mixture is a chemiluminescent solution used to detect and quantify proteins in Western blot analysis. The mixture contains the necessary reagents to generate a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
5 protocols using ecl mixture
1
STAT3 Immunoprecipitation and Western Blot
2
Western Blot Characterization of Protein Samples
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Harvested cells were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl/pH 7.4; 1% NP-40; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF; 1 mM okadaic acid; and 1 mg/ml aprotinin, leupeptin, and pepstatin). Individual samples (30 μg protein) were separated on 8-10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in a PBST solution with 5% fat-free milk for 1 hr at room temperature, and then the membranes were incubated with appropriate dilutions of specific primary antibodies overnight at 4°C. After washing, the blots were incubated with HRP conjugated anti-rabbit or anti-mouse IgG for 1 hr. The blots were developed in ECL mixture (Vector Lab, Burlingame, CA) and visualized by Imager.
3
Immunoprecipitation and Western Blotting Protocol
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LNCaP and C4-2 cells were harvested and lysed in lysis buffer containing 150 mM KCl, 75 mM Hepes (pH 7.5), 1.5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 0.075% NP-40 supplemented with a protease inhibitor cocktail from Roche (USA). The extracted proteins were precleared using a mixture of protein A–Sepharose (CL-4B; GE Healthcare) and antibody overnight at 4°C. Immunoprecipitates were washed with lysis buffer, resuspended in sample buffer, boiled, and analyzed by SDS-PAGE. Individual samples (40 μg of protein) were separated on an 8% SDS polyacrylamide gel and transferred to PVDF membranes from Millipore (Billerica, MA). The membranes were blocked in a PBS-Tween20 solution with 5% fat-free milk for one hour at room temperature before being incubated overnight at 4°C with appropriate dilutions of specific primary AR or TNIK antibodies. After washing, the blots were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG for one hour. Finally, the blots were developed using an ECL mixture from Vector Laboratories (Burlingame, CA) and visualized by Imager.
4
Protein Extraction and Western Blot Analysis
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RIPA protein lysis buffer (Solarbio, Cat#R0020, Beijing, China) and protease inhibitor PMSF were used to extract total cell protein. The volume ratio of RIPA and PMSF is 100 μl:1 μl. We used the Bradford to measure the protein concentration at 595 nm. As for gel prepare, we used the SDS-PAGE Gel Kit (Solarbio, P1200-50T, China) to prepare 10% gel according to its manuscription. The prepared protein sample is added to the gel channel for constant voltage electrophoresis. After the electrophoresis, the protein in the SDS-PAGE gel is transferred to the PVDF membrane. Subsequently, the PVDF membrane was placed in 5% fat-free milk and blocked for 1 h at room temperature. Then the appropriate diluted primary antibody was used to incubate the PVDF membrane at 4 °C overnight. On the second day, wash the PVDF membrane three times with TBST for 10 min each time. After washing, the blots were incubated with HRP conjugated anti rabbit or antimice IgG for 1 h. The blots were developed in ECL mixture (Vector Laboratories, Burlingame, CA) and visualized by Imager. The corresponding antibody and dilution ratio are shown in the list A.
5
Immunoprecipitation and Western Blot Analysis
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LNCaP and C4-2 cells were harvested and lysed in lysis buffer (150 mM KCl,75 mM Hepes, pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 0.075% NP-40 supplemented with protease inhibitor cocktail[Roche,USA]. Extract proteins were precleared using a mixture of protein A-Sepharose (CL-4B; GE Healthcare) and antibody for overnight hr at 4˚C. Immunoprecipitates were washed with lysis buffer and resuspended in sample buffer, boiled and analyzed by SDS-PAGE. Individual samples (40 µg of protein) were separated on 8% SDS polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked in a PBS-Tween 20 solution with 5% fat-free milk for 1 h at room temperature, and then the membranes were incubated with appropriate dilutions of speci c primary AR or TNIK antibodies overnight at 4 °C. After washing, the blots were incubated with HRP conjugated antirabbit or anti-mouse IgG for 1 h. The blots were developed in ECL mixture(Vector Laboratories, Burlingame, CA) and visualized by Imager.
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