Cells were lysed in Cell Signaling lysis buffer (#9803) containing 1mM PMSF (Sigma P7626), 2mM NaF (Sigma S-1504), 2mM Na3VO4 (Sigma S-6508) and 1x protease inhibitor cocktail (Roche 5892953001). 50μg total protein/sample of total lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore IPVH00010). The membranes were then probed with appropriate primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. The blots were developed with HyGLO chemiluminescence reagent (Denville Scientific #E2400) and exposed to HyBlot CL autoradiography film (Denville Scientific #E3012), which was scanned with a flat-bed scanner (UMAX Technologies, Hsinchu, Taiwan). Band densitometry analysis was performed using the NIH ImageJ program. Protein expression was normalized to either Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (Cell Signaling, #2118S) or β-actin (Sigma #A5441) expression.
Hyglo chemiluminescence reagent
Manufactured by Thomas Scientific
Hyglo chemiluminescence reagent is a laboratory chemical used to produce luminescence through a chemical reaction. It is designed for use in various analytical and detection applications in scientific research and testing.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Lysis buffer, by Merck Group (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Na3vo4, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ecl machine, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Hrp conjugated goat secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated goat secondary antibodies, by Thermo Fisher Scientific (1 mentions)
5 protocols using hyglo chemiluminescence reagent
1
Western Blot Analysis of Protein Expression
2
Immunoblotting Protein Detection Protocol
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For immunoblotting, cells were washed with ice-cold phosphate-buffered saline (PBS), lysed in lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1 mM EDTA, and 2% Triton X-100), supplemented with a phosphatase inhibitor mix (Pierce) and a complete protease inhibitor cocktail (Roche). Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk, and probed with the indicated antibodies. Horseradish peroxidase-conjugated goat secondary antibodies were used (1:5000, Invitrogen). Immunodetection was achieved with the Hyglo chemiluminescence reagent (Denville Scientific), and detected by a Bio-Rad ECL machine.
3
Protein Extraction and Western Blot Analysis
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Protein was extracted from cells or mouse liver tissues with lysis buffer (P0013, Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with protease inhibitor cocktail (04693116001; Roche Diagnostics, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (78420; Thermo Scientific, Waltham, MA), and the samples were then centrifuged. Immunoblotting was performed as described previously [3 (link)]. Immunodetection was achieved with Hyglo chemiluminescence reagent (Denville Scientific) and detected by a Bio-Rad ECL machine (Bio-Rad). After signal quantification by densitometry, the results were expressed as a ratio to the loading control in densitometry units.
4
Immunoblotting and Co-Immunoprecipitation Protocols
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For immunoblotting, polypeptides were resolved by SDS–PAGE and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% non-fat dry milk, and probed with the indicated antibodies. HRP-conjugated goat secondary antibodies were used (1:10,000, Invitrogen). Immunodetection was achieved with the Hyglo chemiluminescence reagent (Denville Scientific), and detected by a Fuji ECL machine (LAS-3000). For co-immunoprecipitation, cells were lysed in 1% NP40 lysis buffer (25 mM Tris pH 7.5; 300 mM NaCl, 1 mM EDTA, 1% NP40), supplemented with a complete protease inhibitor cocktail (Roche). After preclearing with protein A/G agarose beads for 1 hr at 4 °C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1–4 μg commercial antibody was added to cell lysate, which was incubated at 4 °C for 8–12 h. After addition of protein A/G agarose beads, incubation was continued for another 2 h. Immunoprecipitates were extensively washed with NP40 lysis buffer and eluted with SDS–PAGE loading buffer by boiling for 5 min before resolution by SDS–PAGE.
5
Immunoblotting and Co-Immunoprecipitation Protocols
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