Radio Immunoprecipitation Assay lysis buffer was employed to lyse 16HBE cells. Next, the collected protein sample was isolated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked by 5% skim milk powder for 1 h. Next, primary antibodies were added into the membranes at 4°C overnight. Primary antibodies include antibodies against B cell leukemia/lymphoma 2 (abbreviated as Bcl-2; ab182858, Abcam), cleaved-caspase-3 (ab32042, Abcam), Bcl2 associated X, apoptosis regulator (abbreviated as Bax; ab32503, Abcam), CDKN1B (ab32034, Abcam) and GAPDH (ab8245, Abcam). After the membranes were incubated with the secondary antibodies at 37°C for 1 h, an ECL Plus reagent (Applygen Technologies Inc., Beijing, China) was employed to develop the bands.
Ecl plus reagent
Manufactured by Applygen
Sourced in China
The ECL Plus reagent is a chemiluminescent detection system used in immunoassays and Western blotting techniques to visualize and quantify target proteins. The reagent produces a luminescent signal when it reacts with the enzyme labels on the target proteins, allowing for their detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab32034, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Protease inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
M iggκ bp hrp secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Merck Group (1 mentions)
Mouse monoclonal anti cxcr4, by Abcam (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by OriGene (1 mentions)
Ab210498, by Abcam (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Alpha imager 5500, by Bio-Techne (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
8 protocols using ecl plus reagent
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Protein Samples
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Cells were lysed in RIPA buffer (Cell Signaling Technology, USA) supplemented with protease inhibitors cocktail (Thermo Fisher Scientific). 50μg extracted protein was separated by SDS-PAGE and transferred to PVDF membranes (BioRad Laboratories, USA). The membrane was blocked with 5% non-fat milk in TBST (10mM Tris, 150mM NaCl, 0.05%Tween 20, pH 8.3) for 1h and then probed with the indicated primary antibodies (Supplementary Table S1) at 4°C overnight with gentle shaking. The following day, the membrane was washed with TBST (5min × 3) and then incubated in m-IgGκ BP-HRP secondary antibody (Santa Cruz, sc-516102) for 1h at room temperature. Then the immunoreactive protein bands were detected using the ECL Plus reagent (Applygen Technologies, Inc., Beijing, China).
3
Western Blot Analysis of CXCR4 and EGFR
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Western blot analysis was performed as reported previously (24 (link)). Cells were lysed in radioimmunoprecipitation assay lysis buffer (EMD Millipore, Billerica, MA, USA). Total protein concentration was determined using a Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (25 (link)). Extracted protein (~30 µg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel, and was then transferred to a polyvinylidene difluoride membrane (EMD Millipore Corporation, Billerica, MA, USA) at 80 V for 2 h at 4°C. The membrane was blocked in skim milk for 1 h, and subsequently incubated overnight at 4°C with a mouse monoclonal anti-CXCR4 (Abcam; dilution 1:2,000; #ab58176) or a rabbit monoclonal anti-EGFR (Cell Signaling Technology, Inc., Danvers, MA, USA; dilution 1:3,000; #4405) antibody. The following day, the membrane was gently washed in Tris-buffered saline with 0.1% Tween-20 (TBST), and then incubated with goat anti-mouse (#ZB-2305) or goat anti-rabbit (#ZB-2301) horseradish peroxidase-conjugated secondary antibodies (OriGene Technologies, Inc.; dilution 1:2,000) for 2 h at room temperature. Subsequent to washing in TBST, the immunocomplexes were detected using ECL Plus reagent (Applygen Technologies, Inc., Beijing, China) (16 (link),26 (link)).
4
Abrin-a Immunoblotting Detection
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Firstly, 100 ng of abrin-a was loaded and separated on 10% SDS polyacrylamide gels under reducing or nonreducing conditions, before transferring onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% BSA, the membranes were incubated with 10D8 antibody diluted to 1:1000 and HRP-labeled anti-mouse secondary antibody (Abcam, Cambridge, UK) diluted to 1:5000. The immunostaining signal was visualized using ECL Plus reagent (Applygen, Beijing, China) and imaged with FluorChem E (ProteinSimple, San Jose, CA, USA).
5
Protein Expression and Apoptosis Analysis
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Tissues and cells were lysed by the RIPA lysis buffer. Protein samples were isolated by SDS-PAGE and then moved to PVDF membranes. Next, the samples were covered with 5% nonfat milk powder for 1 h. Primary antibodies (Abcam, Cambridge, UK) against PACS1 (ab208171, 1:1,000), Bax (ab32503, 1:1,000), Bcl-2 (ab196495, 1:500), cleaved caspase-3 (ab214430, 1:5,000), and GAPDH (ab8245, 1:2,000) were incubated with the membranes overnight at 4°C. After secondary antibodies were incubated with the membranes at 37°C for 1 h, an ECL Plus reagent (Applygen Technologies Inc., Beijing, China) was employed to develop the bands. ImageJ software (National Institutes of Health, Bethesda, MA, USA) was utilized to quantify the result of western blot. GAPDH was set as a loading control. To assess cell apoptosis, apoptosis ratio was measured by the ratio of Bcl-2/Bax protein level.
6
Protein Expression Analysis by Western Blot
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Cells were lysed by radioimmunoprecipitation assay lysis buffer. Protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked by 5% nonfat milk powder for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies include antibodies against Beclin-1 (ab210498, Abcam), LC3 (ab192890, Abcam), glutathione peroxidase 4 (GPX4; ab125066, Abcam) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab8245, Abcam). Membranes were incubated with the secondary antibodies at 37°C for 1 h, and the protein bands were visualized by an ECL Plus reagent (Applygen Technologies Inc).
7
Western Blot Protein Analysis
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Cells were lysed after the indicated treatments. Approximately, 5 mg of total protein for each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk and incubated with appropriate primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The immunostaining signals were visualized using the ECL plus reagent (Applygen, Beijing, China), imaged, and analyzed with an Alpha Imager 5500 (Alpha Innotech, San Leandro, CA).
8
Western Blot Analysis of Cellular Proteins
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The total cellular proteins were lysed in RIPA buffer (Cell Signaling Technology, Inc., MA, USA) supplemented with protease inhibitors. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Extracted protein (30–50 μg) was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (EMD Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h and then probed with the indicated primary antibodies (Supplementary Table S4 ) at 4 °C overnight with gentle shaking. The following day, the membrane was washed with TBST (5 min × 3) and then incubated in secondary antibody (Supplementary Table S5 ) for 2 h at room temperature. Then the immunocomplexes were detected using the ECL Plus reagent (Applygen Technologies, Inc., Beijing, China).
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