Equal amounts of total proteins were separated on a 12% SDS-polyacrylamide gel, and then electro-blotted onto a nitrocellulose membrane. The blots were blocked with 5% dry milk in TBS buffer, and probed with rabbit anti-Hfq, rabbit anti-Crc or rabbit anti-E. coli-S1 antibodies (Sonnleitner and Bläsi, 2014 (link)). Immunodetection of ribosomal protein S1 served as a loading control. The antibody–antigen complexes were visualized with horseradish peroxidase (HRP) conjugated anti-rabbit antibodies (Cell Signaling Technology) using the SuperSignalTM West Pico PLUS chemiluminescent substrate kit (Thermo Scientific). The signals were detected with ChemiDocTM Touch Imaging System (BioRad) and analyzed with ImageLab 5.2.1 (BioRad).
Supersignal west pico plus chemiluminescent substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperSignal™ West Pico PLUS Chemiluminescent Substrate kit is a reagent used for the detection of proteins in western blotting applications. It produces a chemiluminescent signal that can be detected by imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
β actin sc 47778hrp, by Santa Cruz Biotechnology (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Jetprime reagent, by Polyplus Transfection (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (2 mentions)
Smo antibody e 5, by Santa Cruz Biotechnology (2 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Anti flag a8592, by Merck Group (1 mentions)
43 protocols using supersignal west pico plus chemiluminescent substrate kit
1
Hfq, Crc, and S1 Protein Detection
2
Comprehensive Western Blot Procedure
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Western blot analysis was performed according to the standard procedure. Anti‐hTERT (ab32020) and anti‐DKC1 were purchased from Abcam. Anti‐Flag (A8592) was obtained from Sigma‐Aldrich; anti‐PES1 (sc‐166300) and β‐actin (sc‐47778HRP) were purchased from Santa Cruz Biotechnology; anti‐hTERT (abx120550) was purchased from Abbexa. Samples were separated on SDS–polyacrylamide gels and transferred to NC membranes. The membranes were incubated with corresponding antibodies. Immunoreactive proteins were visualized using the ChemiDocTM Imaging System (Bio‐Rad) with a Super SignalTM West Pico PLUS Chemiluminescent Substrate Kit (Thermo). Gray value was analyzed using Scion Image software, and the effect was judged by comparing the ratio of two groups.
3
Protein Extraction and Western Blot Analysis
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Human and mouse keratinocytes were disrupted in M-PER lysis buffer (Thermo Fisher Scientific, #78501) containing protease inhibitors (Thermo Fisher Scientific, #87786) using ultrasonic homogenizer (20% power with 5 times every 2 s) to extract proteins. Then, protein concentrations were determined by BCA method (Thermo Fisher Scientific, #23225). Western blot procedures were followed by the protocol of NuPAGE system (Thermo Fisher Scientific). Briefly, 20 µg of protein samples were loaded for electrophoresis and transferred to polyvinylidene di-fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). After blocking for at least an hour in 5% nonfat dry milk, the membrane was incubated with primary antibodies with appropriate dilutions (Supplementary table 2 ) at 4 °C overnight and followed by incubation with secondary antibodies for 1 h at room temperature. Protein amounts were normalized to rabbit polyclonal anti-human β-actin antibody (1:1000 dilution) (Cell Signaling Technology). Finally, proteins were visualized using SuperSignalTM West Pico PLUS chemiluminescent substrate kit (Thermo Fisher Scientific, #34577) and saved as image files using ChemiDoc XRS+ (Bio-Rad). The signal intensity of protein was quantified using Image LabTM software (Bio-Rad). Original images of representative blots are provided in the Source data file.
4
Mitochondrial Protein Characterization of Maize Dek Mutant
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Crude mitochondria were extracted from 15 DAP kernels of dek504-ref and WT (without pericarp) according to a previously described method [64 (link)]. For BN-PAGE and NADH dehydrogenase activity of complex I analysis, mitochondrial proteins (100 μg) were solubilized using the NativePAGE™ Sample Prep Kit (Invitrogen, Carlsbad, CA), as described previously [64 (link)].
Mitochondrial proteins (20 μg) extracted from WT and dek504-ref kernels at 15 DAP were used for Western blotting. Proteins were separated by SDS–PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated overnight at 4 °C with various target protein antibodies. Following incubation with the HRP-conjugated secondary antibody, the signals were visualized using the SuperSignalTM West Pico PLUS Chemiluminescent Substrate Kit (Thermo, Waltham, MA, USA) in accordance with the instructions of the manufacturer. The antibody dilutions used were 1:1000 for Cox2 (Agrisera), 1:1000 for NAD9 (PHYTOAB), 1:2000 for Cyt-c (Agrisera), 1:2000 for AOX (PHYTOAB), and 1:1000 for ATPase-B (Agrisera).
Mitochondrial proteins (20 μg) extracted from WT and dek504-ref kernels at 15 DAP were used for Western blotting. Proteins were separated by SDS–PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated overnight at 4 °C with various target protein antibodies. Following incubation with the HRP-conjugated secondary antibody, the signals were visualized using the SuperSignalTM West Pico PLUS Chemiluminescent Substrate Kit (Thermo, Waltham, MA, USA) in accordance with the instructions of the manufacturer. The antibody dilutions used were 1:1000 for Cox2 (Agrisera), 1:1000 for NAD9 (PHYTOAB), 1:2000 for Cyt-c (Agrisera), 1:2000 for AOX (PHYTOAB), and 1:1000 for ATPase-B (Agrisera).
5
Quantifying Muscle Iron Transporters
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Ferritin and iron transporter protein content in muscles was measured by Western blot. Procedures were performed as previously described [27 (link)]. Briefly, 40 μg of muscle proteins were separated by SDS-PAGE, transferred to poly-vinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA), blocked in 5% nonfat dry milk in phosphate-buffered saline with Tween-20 (PBST), washed in PBST, and incubated overnight at +4 °C with primary antibodies against ferritin (1:1000, ab75973, Abcam, Cambridge, UK), TfR (1:1000, ab84036, Abcam, Cambridge, UK), DMT1 (1:1000, ABS983, Sigma-Aldrich, St. Louis, MO, USA), and ferroportin (1:1000, PA5-22993, Invitrogen, Waltham, MA, USA). Then membranes were washed three times in PBST and incubated in goat anti-rabbit secondary antibody (1:3000, conjugated with HRP, 130-65-15, BioRad, Hercules, CA, USA) for 2 h at room temperature. Proteins were visualized with SuperSignalTM West Pico PLUS Chemiluminescent substrate kit (Thermo Fisher Scientific, Waltham, MA, USA), detected with BioRad ChemiDocTM Imaging System, and quantified using ImageLab software (BioRad, Hercules, CA, USA). The results were normalized to the total proteins of Ponceau S staining.
6
FLAG-Tagged Protein Western Blotting
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For Western Blot analysis, cells were harvested and normalized to the optical density. After centrifugation the pellet was resuspended in water and SDS sample buffer (500 mM Tris pH 6.8, 10% SDS w/v, 20% Glycerol v/v, 0.05% bromophenol blue w/v). The samples were heated to 95°C for 10 min and vigorously mixed on the vortex for at least 1 min. To remove debris, the samples were again centrifuged (10 min, 13,000 rpm) and the supernatant was loaded into wells of the SDS-PAGE gel. After semidry blotting to a PVDF membrane (Roth; 0.45 μm), protein detection was performed using a monoclonal mouse ANTI-FLAG M2-Peroxidase (HRP) antibody from Sigma-Aldrich. The SuperSignalTM West Pico PLUS Chemiluminescent Substrate Kit (Thermo Fisher) was used to develop the western blot signals.
7
Protein Extraction and Western Blot Analysis
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Protein was extracted from each tissue using extraction buffer [50 mM Tris-MES pH 7.5, 50 mM NaCl, 0.5% SDS (v/v)], and protease inhibitor cocktail P9599 (Sigma-Aldrich, St. Louise, MO, United States). The extract was centrifuged at 12000 rpm at 4°C for 30 min. For immunoblotting, protein samples (18 μg total protein) were separated on 12% SDS PAGE gels and transferred to nitrocellulose membranes (BioTraceTM NT nitrocellulose, Mexico) followed by western blot analysis. After blocking non-specific binding with 5% milk, the blot was subsequently incubated with antibodies generated against the indicated proteins and detected using the Super SignalTM West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific, United States).
8
Western Blot Analysis of Mice Tissues
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Mice tissues were extracted by RIPA buffer using MagNA Lyser Green Beads (Roche Life Science, Penzberg, Germany), subjected to SDS-PAGE separation and transferred onto methanol-activated PVDF membranes (Pall Corporation, NY, USA). The membranes were blocked for one hour at room temperature in 5% non-fat milk containing TBST and incubated with the indicated primary antibodies at 4 °C overnight. The blots were washed three times with TBST for 5 min each; subsequently, the blots were incubated with peroxidase-conjugated secondary antibody for one hour at room temperature. After appropriate washing, the blots were developed by SuperSignalTM West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, MA, USA).
9
Quantifying Chkα Gene and Protein Expression
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RNA from cells and snap-frozen tumor tissue was extracted by QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. cDNA was synthesized by iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). A 10× dilution of cDNA sample and Chkα specific primers were used for quantitative real-time PCR analysis with an iCycler real-time PCR detection system (Bio-Rad). The relative Chkα gene expression to an endogenous control hypoxanthine phosphoribosyltransferase 1 (HPRT1) was calculated based on the delta delta Ct method, where relative gene expression was presented as 2−ΔΔCt.
Total protein from tumor tissue was extracted with a RIPA (Radio-immunoprecipitation assay) buffer with various protease and phosphatase inhibitors to prevent degradation of the protein [28 (link)]. For protein electrophoresis and immunoblotting analysis, 7.5% SDS-PAGE gel was used. A customized rabbit polyclonal primary antibody against Chkα, and an anti-GAPDH antibody (mouse monoclonal, Sigma, St. Louis, MO, USA) were used. Immunoblots were developed by a SuperSignal™ West Pico PLUS Chemiluminescent Substrate kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.
Total protein from tumor tissue was extracted with a RIPA (Radio-immunoprecipitation assay) buffer with various protease and phosphatase inhibitors to prevent degradation of the protein [28 (link)]. For protein electrophoresis and immunoblotting analysis, 7.5% SDS-PAGE gel was used. A customized rabbit polyclonal primary antibody against Chkα, and an anti-GAPDH antibody (mouse monoclonal, Sigma, St. Louis, MO, USA) were used. Immunoblots were developed by a SuperSignal™ West Pico PLUS Chemiluminescent Substrate kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.
10
Western Blot Detection of Starch Metabolism Proteins
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Blots were incubated for one hour in blocking TBST solution (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) Tween 20) containing 3% (w/v) milk powder and then with primary antibodies (against AtGWD, AtPWD and AtSEX4,) diluted 1:1000 (v/v) in blocking solution. After one hour, the primary antibody solution was removed and the membranes were washed for five minutes with TBST buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20). This washing step was repeated six times. Later, blot membranes were incubated with secondary antibody (anti-mouse or anti-rabbit conjugated to alkaline phosphatase or horseradish peroxidase (HRP)) for one hour. The membranes were washed again with TBST buffer as mentioned. To visualize alkaline phosphatase activity, BCIP®®/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) reagent (Sigma-Aldrich) was used following the manual instructions. For HRP conjugated antibodies, signal was detected using the Supersignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher, Waltham, MA, USA) following the manual instructions.
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