Immunoblotting has been described previously [10 (link),11 (link)]. A bicinchoninic acid (BCA; Thermo Fisher Scientific, Waltham, MA, USA) protein assay using bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) as a standard was used to determine protein concentrations. Hippocampal protein samples were heated for 5 min to 95 °C (GLT-1, actin, CP13, MC1, and PHF1) or not heated (vGLUT1, PSD95, Tau-5, HT7, synaptophysin, actin, GSK3β, pGSK3β) and loaded on 10% hand-cast sodium dodecyl sulfate (SDS)-page gels. After transfer, membranes were blocked for 1 h at room temperature (∼23 °C) and then incubated with a primary antibody (see Table 1 for list of antibodies) directed against the protein of interest overnight at 4 °C. The next day, membranes were incubated with the appropriate biotinylated or horseradish peroxidase conjugated secondary antibody for 1 h at room temperature (∼23 °C). Blots were then incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) or Novex AP chemiluminescent substrate (Invitrogen) for 5 min and visualized using Fluorchem E imager (Cell Biosciences, Preston VIC, Australia).
Fluorcheme imager
Manufactured by Cell Biosciences
Sourced in United States
The FluorChemE imager is a laboratory instrument used for the detection and analysis of fluorescent signals. It is designed to capture and quantify the intensity of fluorescent molecules within a sample. The core function of the FluorChemE imager is to provide users with accurate and reliable data on the presence and abundance of fluorescent targets in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (3 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Ab25901, by Abcam (2 mentions)
Pvdf microporous membrane, by Merck Group (2 mentions)
Ab4052, by Abcam (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab62557, by Abcam (2 mentions)
Ab56416, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Protease inhibitor, by Applygen (2 mentions)
Ripa lysis buffer, by Applygen (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by CoWin Biotech (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Novex ap chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rabbit anti beclin 1, by Cell Signaling Technology (1 mentions)
14 protocols using fluorcheme imager
1
Immunoblotting Procedure for Hippocampal Protein Analysis
2
Quantitative Immunoblotting for Cellular Signaling
3
Protein Expression Analysis by Western Blot
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The cells were lysed in Laemmli sample buffer (Bio-Rad; Hercules, CA), and the protein concentrations were determined with a BCA assay (Fisher Scientific). Equal amounts of protein (50 μg) from cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Millipore; Billerica, MA). The membranes were blocked in TBST with 5% non-fat milk and then incubated with the primary antibodies at appropriate dilutions at 4°C overnight. Membranes were washed with TBST solution, followed by incubation with horseradish peroxidase-linked secondary antibody (1:3000) in TBST with 5% non-fat milk for 1 hour at room temperature. Finally, chemiluminescence was detected using the Super Signal West Dura Detection System (Fisher Scientific) and the protein bands were visualized with Cell Biosciences FluorChemE imager (Santa Clara, CA).
4
Western Blot Analysis of Stem Cell Markers
5
Western Blot Analysis of Ganetespib and Lapatinib Effects
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For Western blot analyses, cells were treated with the indicated concentrations of ganetespib and/or lapatinib for 16 hours. Then, the cells were collected and lysed using NP40-based lysis buffer. The protein concentration of each sample was determined using a BCA assay (Thermo Scientific). Equal amounts of protein (50 μg) from each sample were loaded for SDS-PAGE and the separated proteins were transferred to nitrocellulose membranes. The membranes were washed in TBST and blocked in 5% non-fat milk for 2 hours. Then, the membranes were washed in TBST and incubated overnight in the indicated primary antibodies at 4 °C. The membranes were washed again in TBST, followed by incubation in the appropriate species-specific secondary antibody for 1 hour at room temperature. The protein signals were enhanced with ECL solution (Thermo Scientific) and the bands were imaged using a FluorChemE imager (Cell Biosciences; Santa Clara, CA).
6
Quantifying Protein Expression using Western Blot
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Total protein content isolated using the RNeasy PowerLyzer Tissue and Cells kit (see Section 2.10 ) was quantified using the 2-D Quant Kit from GE Healthcare (Pittsburgh, PA, USA) according to the manufacturer’s instructions. Western blot analysis was performed as previously described [46 (link)]. Denatured proteins were subjected to electrophoresis in a 7.5–15% gel. Proteins transferred to a nitrocellulose membrane and incubated with primary antibodies (1:1000) overnight. Membranes were then incubated in secondary antibody (1:2000) for 2 h and exposed in a Cell Biosciences FluorChem E imager (Santa Clara, CA, USA). ImageJ software was used to quantify expression.
7
Phenformin Treatment on Cell Colony Formation
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Cells in the logarithmic phase of growth were collected for inoculation and were seeded (1×103 cells/well) in 6-well plates. Then the cells were treated with phenformin at indicated concentrations for 2 weeks. The medium was changed once on the 3rd day during the 2 week treatment. Following the phenformin treatment, the culture dishes were washed twice with phosphate buffered saline (PBS), fixed with acetone/methanol (1:1) for 5 minutes, and incubated with 0.5% crystal violet for 20 minutes at room temperature. After aspirating the excess stain, the plates were washed with deionized water and air-dried. The colonies were imaged with a FluorChemE imager (Cell Biosciences; Santa Clara, CA) and analyzed with AlphaView software.
8
Western Blot Analysis of Renalase
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As described in previous studies [15 (link), 16 (link)], total tissues were lysed using RIPA buffer, and the protein concentration was determined with a BCA protein assay kit (Pierce Company, Rockford, IL, USA). Protein extracts were used for SDS-PAGE (Invitrogen, Carlsbad, CA, USA), and the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was incubated with primary antibodies (renalase GTX89570, GeneTex, diluted 1 : 1000) overnight at 4°C. After incubation with secondary antibodies (goat IgG antibody GTX228416-01, GeneTex, diluted 1 : 5000) for 1 h at room temperature, the membranes were treated with ECL reagents (170-5061, Bio-Rad, Hercules, CA, USA) prior to visualization using a FluorChem E imager (Cell Biosciences, San Jose, CA) according to the manufacturer's instructions. The specific protein expression levels were normalized to β-tubulin on the same nitrocellulose membrane.
9
N-Glycosidase F Digestion of Xenopus Oocytes
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Xenopus laevis oocytes were injected with cRNA of noninactivating (Δ6–46) Shaker KV channels (Hoshi et al., 1990 (link)) that contained a c-myc epitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) inserted at the C terminus (after Val638) and incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.6) at 17°C. 24 h after injection, oocytes were exposed to 10 mg/ml PNGase F diluted 1:10 in ND96. The enzyme activity was monitored by removing 10 oocytes at different times, supplemented with 100 mM glycine and lysed in 200 µl buffer H (1% Triton X-100, 100 mM NaCl, and 20 mM Tris-HCl, pH 7.4). Lysates were rocked at room temperature for 15 min and then centrifuged at 13,000 rpm for 3 min. The pellet was discarded, and the supernatant was analyzed by Western blot by using anti–Myc mouse monoclonal antibody at a dilution of 1:5,000 (Clontech) and detected with a secondary, goat anti–mouse antibody conjugated to horseradish peroxidase at a dilution of 1:10,000 (Pierce). Membranes were developed by SuperSignal WestFemto (Thermo Fisher Scientific) and visualized by chemiluminescence by using a FluorChem E Imager (Cell Biosciences).
10
Protein Expression Profiling of Hypoxic H9C2 Cells
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Standard western blotting was conducted for protein expression assays in hypoxic H9C2 cells transfected with miR-210 mimic, inhibitor, and NC. Two days following transfection, proteins were isolated with RIPA lysis buffer containing 1 mg protease inhibitors (Applygen Technologies, Beijing, China). The protein contents were quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech, Beijing, China). Primary antibodies against light chain 3 (LC3; ab48394), p62 (ab56416), Caspase8ap2 (ab4052), cleaved caspase 8 (ab25901), cleaved caspase 3 (ab13847), Beclin-1 (ab62557), and the internal controls β-actin and GAPDH were from Abcam (Cambridge, UK). Furthermore, secondary antibodies labelled with horseradish peroxidase were incubated with membranes for 1 h at 37°C. Samples were then transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). Blots were imaged by a FluorChem E imager (Cell Biosciences, San Jose, CA, USA). The grey value of each band was determined with ImageJ version 1.51.
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