Transfection was performed using TurboFect(R0531, Thermo Scientific) following the manufacturer's instructions. After 48h harvested, cells were lysed in HEPES lysis buffer (20 mM HEPES, pH 7.2, 50 MmNaCl, 0.5% Triton X-100, 1 mMNaF and 1 mM DTT) supplemented with protease inhibitor cocktail (Roche). Immunoprecipitations were performed using the indicated primary antibody for 3-4h and protein A/G-agarose beads (Santa Cruz) overnight at 4°C. The resulting immunoprecipitates were washed at least three times in HEPES lysis buffer. Lysates and immunoprecipitates were examined using the indicated primary antibodies followed by detection with the related secondary antibody and the SuperSignalchemiluminescence kit (Thermo).
Supersignal chemiluminescence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium
The SuperSignal chemiluminescence kit is a laboratory reagent designed to detect and quantify proteins in Western blot analyses. It utilizes a chemiluminescent substrate to generate a luminescent signal that can be captured and measured, providing a sensitive and reliable method for protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by GE Healthcare (5 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (4 mentions)
Image lab, by Bio-Rad (3 mentions)
Phosphatase inhibitor, by Merck Group (3 mentions)
Biomax mr x ray film, by Kodak (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Resolving polyacrylamide gradient gels, by Bio-Rad (2 mentions)
Ne per reagent, by Thermo Fisher Scientific (2 mentions)
Bradford dye based assay, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Vecad, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Agfa x ray film, by AGFA HealthCare (2 mentions)
Pro pre protein extraction solution, by iNtRON Biotechnology (2 mentions)
Horseradish peroxidase labeled anti mouse antibodies, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ls b3384, by LSBio (2 mentions)
39 protocols using supersignal chemiluminescence kit
1
Immunoprecipitation and Western Blotting
2
Protein Interaction Identification by IP-Western
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Cell lysates were prepared in RIPA buffer with protease inhibitor cocktail. For each immunoprecipitation assay, 2 mg protein was used, and 2 µg of indicated antibodies was added for each reaction. Isotype-matched IgG was used as a negative control. Antibodies were mixed with equal amounts of protein lysate and incubated at 4°C overnight with rotation. Lysates were incubated with 50 µl of 50% protein A or G agarose (Santa Cruz Biotechnology, Inc.) with rotation for 3 h. Then beads were washed with RIPA buffer three times. Immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting. Transfer membranes were probed with indicated primary and secondary antibodies. The membranes were analyzed with the Super Signal chemiluminescence kit (Thermo Fisher Scientific). ImageJ was used for quantification analysis of the band density of target proteins. All of the immunoprecipitation and Western blots were repeated at least three times.
3
HEK293T Cell Lysis and Immunoprecipitation
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At 36 h after the transfection, HEK293T were lysed in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1mM NaF, 1mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10mM NaF and 1mM Na3VO4). Immunoprecipitations were performed using anti-Flag or anti-Myc antibody and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C. PLEKHO1 and TRAF2, including full-length proteins and truncated mutants, in lysates or immunoprecipitates were examined by anti-Myc (Cell Signaling Technology) and anti-Flag (Sigma-Aldrich, St. Louis, MO, USA) primary antibodies, respectively, and the appropriate secondary antibodies in immunoblotting, followed by detection with SuperSignal chemiluminescence kit (Thermo Fisher Scientific) [6 (link),7 (link),19 (link)].
4
Immunoblot Analysis of ZNF750 Protein
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For immunoblot analysis, proteins were extracted with RIPA buffer containing cocktail inhibitors (Roche), separated on SDS polyacrylamide gels and then transferred onto nitrocellulose membranes (GE Healthcare) by a wet-transfer system. Membranes were blocked with TBS-0.1% Tween and 10% milk and incubated overnight with primary antibodies. The following day, membranes were washed and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were visualized with the Super Signal chemiluminescence kit (Thermo Scientific). The following antibodies were used: anti-ZNF750 (1:1000; Sigma HPA023012) and anti-β-actin (1:50,000; Sigma A5441).
5
Western Blot Analysis of Cellular Proteins
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The cells were lysed in 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl2, 10 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5 mM EDTA and protease inhibitors, separated on SDS-PAGE and transferred to nitrocellulose sheets at 400 mA for 2 h at 4 °C. Western blot analysis was performed using antibodies against TRPM7 (Bethyl, Montgomery, AL, USA), MagT1 (Abcam, Cambridge, UK), SLC41A1, Mrs2, cyclophilin D (CypD), thioredoxin-interacting protein (TXNIP), paraoxonase 2 (PON2) and superoxide dismutase 2 (SOD2) (Thermo Fisher Scientific, Waltham, MA, USA), heat shock protein (Hsp) 27 (Cell Signaling Technology, Danvers, MA, USA), Hsp70 and actin (Santa Cruz Biotechnology, Dallas, TX, USA). After extensive washing, secondary antibodies labeled with horseradish peroxidase (GE Healthcare, Waukesha, WI, USA) were used. Immunoreactive proteins were detected by the SuperSignal Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA). All the experiments were performed three times, and a representative blot is shown. Densitometry of the bands was performed with the software ImageLab (Bio-Rad, Hercules, CA, USA) on three blots ± (SD).
6
Western Blot Analysis of Protein Expression
7
Western Blot Analysis of Protein Levels
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Total cell lysates were prepared and western blotting was performed as described previously 31 . Proteins (20–40 μg) were separated by SDS–PAGE on 4–10% resolving polyacrylamide gradient gels (Bio‐Rad, Hercules, CA) and electroblotted to PVDF membranes. The membranes were incubated with specific primary antibodies, followed by 1 h incubation with appropriate peroxidase‐coupled secondary antibodies. Signals were detected using a SuperSignal chemiluminescence kit (Thermo Fisher, Rocksord, IL) and images were captured using ImageLab software (Bio‐Rad). Band intensity was quantified using the Image J software. Separate aliquots were probed for β‐actin to assess loading. Protein levels were expressed as relative expression/ β‐actin (mean ± SD).
8
Immunoblotting Analysis of Protein Expression
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Total proteins were extracted from the cells using RIPA buffer (Keygen) with 1 mM proteinase inhibitor PMSF (Keygen) and cocktail (Roche). Forty micrograms of protein were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked for 1 h at room temperature in TBST containing 5% BSA, and then incubated overnight at 4 °C in TBST containing 5% BSA and following antibodies: CUL4B (Sigma, C9995); CD44 (Sigma, HPA005785 or Abcam, ab157107); NOTCH1 (CST, 3608S); NUMB (Abcam, ab4147); MYCN (Genetex, GTX133721); and GAPDH (CST, 5174S). Membranes were washed in TBST, incubated with a secondary antibody and conjugated with horseradish peroxidase for 1 h at room temperature. After washes with TBST, bands were detected using a SuperSignal Chemiluminescence kit (Thermo). Intensity of bands was qualified with GAPDH as the reference by Quantity One software.
9
Immunoprecipitation and Western Blotting
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Cells were transfected with the indicated plasmids and lysed in RIPA lysis buffer [25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5% Glycerol, 1% Triton X-100, 2 mM EDTA, and 1 mM DTT supplemented with protease inhibitor cocktail (Roche)] at 48 h after transfection. Immunoprecipitation was performed by incubation with the indicated primary antibodies for 4 h and protein A/G agarose beads (#20423, Thermo Scientific) overnight at 4 °C. The beads were washed at least three times with RIPA lysis buffer. Lysates and immunoprecipitated sampless were examined by using the indicated primary antibodies followed by the related secondary antibodies and the SuperSignal Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA).
10
Fractionating Cervical Cancer Cells
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