Cells were lysed in Laemmli buffer and proteins were separated by polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF Transfer Membrane (NEN Life Science Products, USA) which was then blocked for 1 hour with 3% dry milk in PBS-0.05% Tween 20 and incubated for 4h with the primary antibody. After 3 washings, membranes were incubated with the secondary horseradish peroxydase-conjugated antibody for 1h, and revealed by chemoluminescence using home-made ECL acquired with the ImageQuant LAS4000 device (GE Healthcare, USA). The membranes were re-probed with anti-ERK1/2 antibodies used as a control for protein loading.
Imagequant las 4000 device
Manufactured by GE Healthcare
Sourced in United States
The ImageQuant LAS 4000 is a laboratory imaging device designed for the analysis of fluorescent and chemiluminescent samples. The core function of this product is to capture and quantify digital images of these samples.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (4 mentions)
Flexigene dna kit, by Qiagen (1 mentions)
Nylon membrane, by Merck Group (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
λ dna hindiii digest, by New England Biolabs (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
5 protocols using imagequant las 4000 device
1
Western Blot Protein Detection
2
CNBP Expanded Alleles Detection
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Genomic DNA was extracted from 500 µl of anticoagulated peripheral blood using a Flexigene DNA Kit (Qiagen, Hilden, Germany) and diluted to a final volume of 25 μl with double-distilled water. The quality and quantity of DNA were assessed using a Denovix spectrophotometer and by 1% agarose gel electrophoresis. CNBP expanded alleles were detected as previously described (Nakamori et al., 2009 (link)), with modifications. Briefly, 2 μg of genomic DNA was digested with AluI and HaeIII and the fragments were resolved by 0.4% agarose gel electrophoresis at 40 V for 40 hr. After denaturation and neutralization, the DNA was transferred to a nylon membrane (MilliporeSigma, Burlington, MA) and fixed by UV cross-linking using a Stratalinker 2400 (Stratagene, San Diego, CA). The membrane was hybridized for 16 hr at 65°C with a digoxigenin (DIG)-labeled locked nucleic acid (LNA) probe (CCTG)5 at a concentration of 10 pmol/ml. After washing at high stringency, the signal was revealed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Basel, Switzerland) and visualized using an ImageQuant LAS 4000 device (GE Healthcare, Chicago, IL). Bands were sized by running two sets of molecular weight markers alongside the samples: DNA Molecular Weight Marker XV (Expand DNA Molecular Weight Marker, Roche) and λ DNA-HindIII Digest (New England Biolabs, Ipswich, MA).
3
Osteoclast Differentiation Protein Analysis
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BMMs were seeded at a density of 1 × 106 cells/well in 6-well plates, followed by stimulation using M-CSF and RANKL with or without Lon of designated dosages for the set times or the specified time for various concentrations. To extract proteins, we utilized the radioimmunoprecipitation (RIPA) lysis buffer to split the treated cells. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to isolate the proteins, which were then deposited to a nitrocellulose membrane (Thermo Fisher Scientific, Shanghai, China). After that, the nitrocellulose membranes were infiltrated in 5% skim milk to block non-specific binding immunoreactivities. Subsequently, the membranes were subjected to incubation overnight at a temperature of 4°C with primary antibodies (1:1,000) and were gently shaken. On the following day, membranes were rinsed 3 times utilizing Tris-buffered saline plus 0.1% Tween 20 (TBST), followed by incubation using corresponding anti-mouse or anti-rabbit antibodies for 1 hour at ambient temperature. The images were captured with the aid of an ImageQuant LAS-4000 device (GE Healthcare, Chicago, Illinois, United States) and processed utilizing the ImageJ.
4
Western Blot Analysis of Cell Proteins
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Proteins were extracted from cultured cells with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF, with protease inhibitors) to perform Western blot analysis. Protein samples were resolved by 6% acrylamide gels and electroblotted onto a PVDF membrane. The membrane was blocked in 3% BSA in tris-buffered saline, 0.1% Tween 20 (TBST) for 1 h, incubated with primary antibody diluted in blocking solution overnight at 4 °C, washed three times with TBST, incubated with secondary antibodies for 1 h at RT, and washed three times with TBST. For the detection of signals, SuperSignal West Femto (ThermoFisher Scientific) was used. Membranes were imaged on an ImageQuant LAS 4000 device (GE Healthcare Life Sciences, Chicago, IL, USA). The primary antibodies and dilutions used were: mouse anti–GAP120 (1:200; Santa Cruz Biotechnology), mouse anti-GAPDH (1:1000; Invitrogen) and rabbit anti-separase (1:200; Abcam). Blots were detected using goat anti-mouse and goat anti-rabbit (1:2500; ThermoFisher Scientific). ImageJ was used for quantification of the signal.
5
Western Blot Analysis of Protein Expression
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Cultured cells were lysed by adding lysis buffer (radioimmunoprecipitation assay to phenylmethane sulfonyl fluoride ratio, 99:1), and protein was extracted in 1X SDS sample buffer following centrifugation at 12,000 × g for 15 min at 4°C. The protein concentration of each sample was measured using a BCA protein assay kit (cat no. ab207002; Abcam). Equal amounts of protein were separated using SDS-PAGE (12%), transferred onto polyvinylidene difluoride membranes, and then blocked in Tris-buffered saline/Tween-20 containing 5% non-fat milk for 1 h at room temperature. The membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-mouse polyclonal p38 (cat no. 8690S; 1:1,000 dilution), p-p38 (cat no. 4511S; 1:1,000 dilution) (both from Cell Signaling Technology, Inc.), FN (cat no. 15613-1-AP; 1:1,000 dilution; ProteinTech Group, Inc.) and GAPDH (cat no. 10494-1-AP; 1:5,000 dilution; ProteinTech Group, Inc.) antibodies; AIF-1 and α-SMA antibodies were used as described earlier. Following incubation with horseradish peroxidase conjugated-secondary antibodies (cat no. ZB2301; 1:2,000 dilution; OriGene Technologies, Inc., Rockville, MD, USA) for 1 h at 37°C, the membranes were treated with an enhanced chemiluminescence substrate kit to obtain visible bands, which were observed with a ImageQuant LAS-4000 device (GE Healthcare, Chicago, IL, USA).
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