Treated rat NP cells were immediately placed on ice and washed with cold PBS. To prepare the total cellular proteins, the cells were lysed with lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, complete protease inhibitor cocktail (Roche, IN, USA), 1 mM NaF, and 1 mM Na3VO4. Heat-denatured samples were separated on sodium dodecyl sulfate polyacrylamide gels and electrotransferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were then blocked with blocking buffer (5% BSA and 0.1% NaN3 in PBS) and subsequently incubated overnight at 4 °C with anti-CCN2 (1:1000, Santa Cruz Biotechnology or Abcam, Cambridge, UK) antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Tokyo, Japan). Chemiluminescent signals were visualized with an Immobilon Western Chemiluminescent HRP Substrate (Millipore) and scanned using an Ez-Capture MG imaging system (ATTO, Tokyo, Japan). The western blot data were quantified using Image J pixel analysis (NIH Image software). Western blot data are presented as band intensity normalized to that of the loading control (β-actin). To measure the band intensity, the data shown are representative of at least three independent experiments.
Can get signal immunoreaction enhancer solution
Manufactured by Toyobo
Sourced in Japan
The Can Get Signal Immunoreaction Enhancer Solution is a laboratory product designed to enhance immunoreactions. It is formulated to improve the visualization and detection of target analytes in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime western blotting detection reagent, by GE Healthcare (8 mentions)
Imagequant las 4000 mini, by GE Healthcare (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Ecl western blotting detection reagent, by GE Healthcare (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Fetal bovine serum fbs, by Nichirei Biosciences (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ecl prime system, by GE Healthcare (3 mentions)
Hrp linked anti rabbit or anti mouse antibody, by GE Healthcare (3 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (3 mentions)
Dulbecco s modified eagle s medium dmem, by Fujifilm (3 mentions)
Anti cd9, by System Biosciences (3 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Can get signal immunoreaction enhancer solution, by GE Healthcare (2 mentions)
Anti rabbit igg horseradish peroxidase, by GE Healthcare (2 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Las 3000, by Fujifilm (2 mentions)
Sample buffer, by Nacalai Tesque (2 mentions)
Chemidoctm mp imaging system, by Bio-Rad (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
67 protocols using can get signal immunoreaction enhancer solution
1
Protein Extraction and Western Blot Analysis
2
Western Blotting of Brain Tissue Proteins
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Western blotting was performed as described previously36 (link). Briefly, hippocampus and cerebral cortex tissues were homogenized in an ice-chilled RIPA Buffer (FUJIFILM Wako Pure Chemical Corporation, Chuo, Osaka, Japan) containing protease inhibitors (cOmplete, Sigma-Aldrich Japan K.K., Meguro, Tokyo, Japan). The lysate was electrophoresed on a 10% Bis–Tris gel in 1× MOPS buffer. After transfer onto polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using ImmunoBlock (DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan). Membranes were incubated for 24 h at 4 °C with primary antibody in Can Get Signal Immunoreaction Enhancer Solution (Toyobo Co., LTD., Kita, Osaka, Japan). Anti-PRKAR1B antibody (PA5-87652, Thermo Fisher Scientific K.K., Minato, Tokyo, Japan) was used at a dilution of 1:2000, and anti-actin antibody (A3853; Sigma-Aldrich, St. Louis, MO, USA) was used at 1:10,000. The secondary antibody was an anti-rabbit IgG horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) and was used at 1:100,000 in Can Get Signal Immunoreaction Enhancer Solution. Signals were developed by ECL Western Blotting Detection Reagents (GE Healthcare) and detected using an ImageQuant LAS 4000 Image Analyzer (GE Healthcare Japan Corporation, Hino, Tokyo, Japan).
3
SDS-PAGE and Western Blotting Analysis
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SDS‐PAGE and subsequent western blotting analyses were performed as previously described.29 Cell lysates were prepared by solubilizing cells in radio‐immunoprecipitation assay (RIPA) buffer (50 mM Tris‐HCl [pH 7.5] containing 150 mM NaCl, 1% NP‐40, and 0.1% sodium deoxycholate) supplemented with protease‐inhibitor and phosphatase‐inhibitor cocktails (Sigma). Samples were then mixed at a 1:4 ration with 5 × sample buffer (300 mM Tris‐HCl [pH 6.8] containing 10% 2‐mercaptoethanol, 10% SDS, 50% glycerol and 0.05% Coomassie Brilliant Blue) and boiled for 5 min. The resulting samples (20 μg protein/lane) were subsequently separated by SDS‐PAGE using pre‐cast 5%–20% Tris‐glycine gradient gels (Atto Corporation, Tokyo, Japan) and transferred to PVDF membranes (Wako Pure Chemical). Membranes were blocked by incubation with TBS containing 0.1% Tween 20 (TBS‐T) and 5% skim milk (Megmilk Snow Bland, Tokyo, Japan) or BSA (Sigma) for 1 h, probed with the appropriate primary antibodies diluted with Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) at 4°C, washed extensively with TBS‐T, and incubated with HRP‐conjugated secondary antibodies (1:5000 dilution) (GE Healthcare, Little Chalfont, UK). Finally, the membranes were washed, developed by incubation with ECL detection reagents (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare).
4
Western Blot Analysis of EHF, STAT3, and ACTB
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Cell lysates were resolved using 12% SDS‐PAGE. Anti‐EHF (Abcom, ab167264), anti‐p‐STAT3 (SAB4300033‐100UG, rabbit polyclonal), anti‐STAT3 (SAB2102317‐100UL, rabbit polyclonal), and anti‐ACTB (ACTN05 [C4]) antibodies were used as primary antibodies. Protein‐blotted membranes were incubated with antibodies using Can Get Signal Immunoreaction Enhancer Solution (Toyobo) at 4℃ overnight for the primary antibodies and at room temperature for 1 hour for secondary antibodies, followed by visualization using the ECL Prime system (GE Healthcare). The protein signals were detected using LAS‐3000 (Fujifilm).
5
Western Blot Analysis of Protein Expression
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Cells were lysed on ice in RIPA buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1%NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 5 mM EDTA) containing protease inhibitors (aprotinin, leupeptin, pepstatin, PMSF) and phosphatase inhibitors (NaF, Na3VO4). After rocking for 1 h at 4 °C, samples were centrifuged at 100,000 x g for 30 min, and the supernatants used as cell lysates. The protein content in cell lysates was estimated with the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Samples containing equal amounts of protein were mixed with 2x Laemmli sample buffer and incubated at 95 °C for 3 min, followed by separation on 9 or 12 % polyacrylamide gels and transfer to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in 5 % non-fat dried milk in phosphate-buffer saline (PBS) containing 0.05 % Tween-20, and probed with primary antibodies, followed by secondary peroxidase-labeled anti-rabbit or mouse IgG. The Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) was occasionally incubated with primary antibodies to enhance immunoreaction. Protein expression was detected with a chemiluminescence reagent (Perkin-Elmer, Boston, MA, USA), and the resulting images examined with a LAS-1000 (Fuji Film, Tokyo, Japan) image analyzer. β-Actin was used as the internal control to normalize the levels of proteins of interest.
6
Protein Extraction and Western Blot Analysis
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Cell lysates were extracted using RIPA buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, .1% SDS and .5% sodium deoxycholate) containing a protease inhibitor cocktail (200 μmol/L AEBSF, 10 μmol/L leupeptin and 1 μmol/L pepstatin A) and phosphatase inhibitor cocktail (10 mmol/L NaF and 1 mmol/L Na3VO4). Protein samples were prepared by mixing lysates with 4 × sample buffer (.25 mol/L Tris‐HCl [pH 6.8], 40% glycerol, 8% SDS, 20% 2‐mercaptoethanol and .02% bromophenol blue) and then boiling at 95°C for 5 minutes. Equal amounts of total protein were fractionated in 5%‐10% SDS‐PAGE, transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, VT, USA), and incubated with primary antibodies in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan). Primary antibody binding was detected using the Pierce Western Blotting Substrate (Thermo Fisher Scientific) with HRP‐conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Signals were visualized using ImageQuant LAS 4000 Mini (GE Healthcare) and quantified with ImageJ software.
7
Western Blot Analysis of A549 Cells
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WB was performed according to the previously reported method.47 (link) Proteins were extracted from A549 cells using a sample buffer (Nacalai Tesque), and the lysates were denatured at 95°C for 3 min. The lysates were then resolved by SDS-PAGE (SuperSep Ace gel; FUJIFILM Wako, Osaka, Japan) and transferred to a polyvinylidene difluoride membrane (Pall Corporation, Port Washington, NY, USA). Antibody reactions were performed using the Can Get Signal immunoreaction enhancer solution (Toyobo, Osaka, Japan). Immunoreactivity was visualized using Chemi-Lumi One Super (Nacalai Tesque) or ImmunoStar LD (FUJIFILM Wako) and ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this study are listed in the Antibody List.
8
Western Blot Analysis of Cell Lysates
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Western blot analysis was performed using the antibodies described above. In brief, total cell lysates were separated by SDS-PAGE (5–20% gels; ATTO, Tokyo, Japan) under reducing conditions and then transferred to a PVDF membrane (PALL, SP, Brazil). Antibody reactions were performed with Can Get Signal® Immunoreaction Enhancer Solution (Toyobo). Peroxidase activities on the membrane were visualized with ECL Western Blotting Detection Reagents (GE Healthcare) or ImmunoStar® LD (Wako Pure Chemical Industries, Osaka, Japan), and a ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA).
9
Lectin Staining with Biotin-Conjugated MAM
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Lectin staining with a biotin-conjugated MAM lectin was performed according to Refs.44 (link),45 (link). The de-paraffinized sections were antigen activated at 90°C for 40 min by HistoVT One (Nacalai Tesque), washed with PBS, and then pretreated with 5% normal goat serum/1% BSA (Cedarlane Laboratory, Ontario, Canada) in PBS for 1 h at room temperature for blocking. Then, the sections were treated with biotin-conjugated MAM lectin (10 μg/mL) diluted in Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) overnight at 4°C. After a washing with PBS, the cells were treated with streptavidin-CY5 (Southern Biotech, 7105-15, 1:1,000 diluted) for 1 h in the dark. The stained cells were examined with a confocal laser scanning imaging system (LSM510, Carl Zeiss).
10
Protein Separation and Western Blot
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Five micrograms of total protein was separated on a precast 10%–20% gradient gel (Funakoshi, #291M) and blotted on a nitrocellulose membrane using a semi-dry blotter following a standard protocol. Antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, NKB-101) and detected with ECL Prime (GE Healthcare, #RPN2232).
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