Western blot analysis was performed by normalization to β-actin or the baseline expression level. Cells were lysed with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail III (Millipore, Billerica, MA). The proteins were separated by electrophoresis using 4-20% or 7.5% SDS-PAGE gel, and transferred onto nitrocellulose membrane. The membranes were incubated with the first antibody, respectively: anti-TOPK antibody (BD Biosciences, San Jose, CA), anti-FOXM1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA (Roche), or anti-β-actin (Sigma-Aldrich). Finally, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody and protein bands were visualized by enhanced chemiluminescence detection reagents (GE Healthcare, Pittsburgh, PA). We generated mouse anti-MELK monoclonal antibodies using partial recombinant MELK protein (264-601 amino acids of MELK) as an immunogen by the methods as described previously [31 (link)].
Enhanced chemiluminescence detection reagent
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
Enhanced chemiluminescence detection reagents are laboratory reagents designed to facilitate the detection and analysis of specific proteins or molecules in a sample. These reagents generate a luminescent signal when they interact with the target analyte, allowing for sensitive and quantitative measurements.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Lysis buffer, by Cell Signaling Technology (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Abcam (2 mentions)
Anti h3k4me3, by Merck Group (2 mentions)
Anti smyd3, by Abcam (2 mentions)
Anti foxp3, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Anti smad3, by Abcam (2 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (2 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (2 mentions)
X ray film, by Fujifilm (2 mentions)
54 protocols using enhanced chemiluminescence detection reagent
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of FIR Protein
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Culture medium was removed, and cells were washed twice with cold (4°C) PBS. Then, cells were lysed with 1:20 β-mercaptoethanol in 2X sample buffer and incubated at 100°C for 5 min. Whole-cell lysates were assayed for protein content (Bio-Rad, Hercules, CA, USA), and 10 μg of proteins were separated by SDS-PAGE on 7.5% or 10–20% XV PANTERA gels before being transferred onto polyvinylidene fluoride membranes using a tank transfer apparatus. The membranes were blocked with 0.5% skimmed milk in PBS overnight at 4°C. Antigens were detected with enhanced chemiluminescence detection reagents (GE Healthcare UK Ltd., Buckinghamshire, UK). Membranes were incubated with primary antibodies for 1 h at room temperature, followed by three 10-min washes with 1XPBS/0.01% Tween 20. Membranes were then incubated with commercial secondary antibodies, followed by three 15-min washes with 1XPBS/0.01% Tween 20. The primary mouse monoclonal antibody against the FIR C-terminus (Total FIRs 6B4) was prepared by Dr. Nozaki [16 (link)].
3
Protein Extraction and Detection
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Total proteins were extracted from ventricular myocardium or cultured NRVMs with 1× sampling buffer (41 mM tris-HCl, 1.2% SDS, and 8% glycerol). Protein concentration was determined with bicinchoninic acid (BCA) reagents (Pierce Biotechnology, Rockford, IL). Equal amounts of proteins were loaded to each lane of the SDS–polyacrylamide gel and fractionated via electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot apparatus (Bio-Rad, Hercules, CA) and immunodetected with anti-PDE1A (1:800; SC-50480, Santa Cruz Biotechnology), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; G8795, Sigma-Aldrich), anti-HA (1:1000; 3724, Cell Signaling), anti-GFP (1:1000; SC-9996, Santa Cruz Biotechnology), anti-tubulin (1:1000; E7-s, DSHB), anti-CryAB (1:1000; ADI-SPA-222, Enzo Life Sciences), anti-Psmb5 [1:1000; custom-made (30 (link))], or anti–p-Rpn6 [1:5000; custom-made (13 )] as the primary antibodies and appropriate horse radish peroxidase–conjugated secondary antibodies. The bound secondary antibodies were detected using the enhanced chemiluminescence detection reagents (GE Healthcare, Piscataway, NJ). Blots were imaged and quantified using the Quantity One or Image Lab software (Bio-Rad). For some of the blots, the total protein content derived from the stain-free protein imaging technology was used as in-lane loading control (8 (link)).
4
Antibody Characterization for BDNF Signaling
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Antibody against tyrosine hydroxylase (TH) was purchased from Merck Millipore (Billerica, MA, United States). Polyclonal rabbit anti-BDNF antibody was purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, United States). Antibodies against TrkB, ERK, phospho-ERK, AKT, phospho-AKT, CREB, phospho-CREB, GAPDH, and Alexa Fluor 594-conjugate anti-rabbit IgG antibody were obtained from Cell Signaling Technology (Boston, MA, United States). Protein assay dye reagent concentrate was purchased from Bio-Rad (Hercules, CA, United States). Enhanced chemiluminescence detection reagents were obtained from GE Healthcare (Uppsala, Sweden). A 3,3-N-diaminobenzidine tetrahydrochloride (DAB) substrate kit was purchased from Dako Corporation (Carpintera, CA, United States). Anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody and MPTP were purchased from Sigma-Aldrich (St. Louis, MO, United States). Mouse anti-BDNF antibody and proteinase inhibitor K252a were bought from Abcam (Cambridge, United Kingdom). Goat-anti Mouse Alexa-568 was obtained from Invitrogen (Carlsbad, CA, United States).
5
Detecting Endogenous TOPK Protein in Glioma Cells
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To detect the endogenous TOPK protein in glioma cells, cells were lysed in lysis buffer; 50 mmol/L Tris-HCl (pH 8.0) containing 150 mmol/L NaCl, 0.5% NP-40 and 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, CA, USA). Cell lysates were prepared as described [18 ]. The amount of total protein was estimated by protein assay kit (Bio-Rad, Hercules, CA, USA), and then proteins were mixed with SDS sample buffer and boiled before loading at 10% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto PVDF membrane (Merck Millipore, Billerica, MA, USA). Membranes, including proteins, were blocked by blocking solution and incubated with anti-PBK/TOPK monoclonal antibody (BD Biosciences, San Jose, CA, USA) for detection of endogenous TOPK protein. Finally, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody and protein bands were visualized by enhanced chemiluminescence detection reagents (GE Healthcare, Buckinghamshire, UK). ß-Actin was examined to serve as a loading control.
6
Immunoblotting of Protein Lysates
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Cell lysates were prepared and immunoblotting was carried out as previously described27 (link). Briefly, 30 μg of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to an Immobilon membrane (Millipore, Bedford, MA, USA) that was blocked and probed with primary antibodies followed by the horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA). Peroxidase-labeled bands were visualized with enhanced chemiluminescence detection reagents (GE Healthcare) according to the manufacturer’s instructions. To confirm equal loading, the membrane was reprobed with anti-β-actin antibody.
7
Western Blot Analysis of EMT Markers
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Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were assayed using a BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.) with bovine serum albumin (Invitrogen) as the standard. 10% SDS-PAGE was used to separate equal amounts of proteins, which were transferred to PVDF membranes. The membranes were blocked with 5% (W/V) nonfat-dry milk in TBST for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies against eIF5A2 (1:1000; Abcam, Cambridge, UK) and GAPDH (1:1000; Abcam), and an EMT antibody sampler kit (1:1000; CST, Danvers, MA). After washing three times with TBST, the membrane was incubated for 2 h at room temperature with peroxidase-conjugated secondary antibodies (1:2000; Abcam). The protein bands were developed using enhanced chemiluminescence detection reagents (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s protocol.
8
Immunoblotting of Mammary Tissue Lysates
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Flash frozen mammary tissues were lysed as in [6 (link)] and 20 μg of protein were separated by SDS-PAGE. Membranes were immunoblotted with the following primary antibodies: E-cadherin (BD Biosciences, Mississauga, ON, Canada; 610182, 1:1,000); Cre recombinase (Novagen/EMD Millipore, Billerica, MA, USA; 69050, 1:1,000); P-EGFR (Y1068) (Cell Signaling/New England Biolabs, Pickering, ON, Canada; #3777, 1:1,000); EGFR (Cell Signaling #2322, 1:1,000); P-ErbB2 (Y1248) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; #12352, 1:500); ErbB2 (Santa Cruz Biotechnology #284, 1:1,000); Hsp90 (Cell Signaling #4874); PyV mT (a generous gift from Dr. S. Dilworth, Ab750, 1:1000), c-Myc (Santa Cruz Biotechnology #764); P-PDGFRβ (Y1021) (Cell Signaling #2227, 1:1,000); PDGFRβ (Cell Signaling #3175, 1:1,000); α-tubulin (Cell Signaling #2125). Horse radish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA. GE Amersham (Bai d'Urfe, QC, Canada) enhanced chemiluminescence detection reagents were used to visualize the immunoblots.
9
Western Blot Analysis of Protein Markers
10
SDS-PAGE Protein Detection and Analysis
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Following separation by glycine- or Tricine-buffered SDS-PAGE76 (link), proteins were detected either by Coomassie Brilliant Blue R250 staining, digital autoradiography or immunoblotting. Dried radioactive gels were exposed to phosphor screens and scanned using the TyphoonTM Trio variable mode imager (GE Healthcare). Analysis and quantification of gel images was performed using the ImageQuant software (Version 5.1, GE Healthcare). For the analysis of experiments by both digital autoradiography and immunoblotting, proteins were first transferred from polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a semi-dry transfer method, before the dried membrane was exposed to a phosphor screen for analysis. The same membrane was then analysed by immunoblotting with the appropriate antisera (directed against human LONM, CLPX, CLPP and NDUFA9) as described previously38 (link). Antiserum against rat OTC, was kindly provided by Nick Hoogenraad (La Trobe University). MDH antibody-horse radish peroxidase (HRP) conjugate (GTX40570) was from GeneTex. In general, membrane bound primary antibodies were detected using HRP-coupled secondary antibodies (anti-mouse or anti-rabbit IgG, Sigma-Aldrich) incubated with enhanced chemiluminescence detection reagents (GE Healthcare) and images captured with a ChemiDoc XRS+ system (Bio-Rad) using Image Lab Software (Bio-Rad).
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