Genomic DNA from cells was extracted using the DNA easy kit according to the manufacturer's instruction and treated with RNase A. Extracted DNA (500 ng) was biotin labeled at the 3′-OH end using the Biotin 3′-end DNA Labeling Kit (Thermofisher Scientific). Biotin-labeled DNA was separated on 0.8% agarose gel and transferred overnight to Zeta-Probe GT membrane (Bio-Rad) by capillary action. DNA on the membrane was UV cross-linked using Spectrolinker XL-1500 UV crosslinker (Spectronics Corporation). Biotin-labeled DNA was detected using chemiluminescent nucleic acid detection kit (Thermofisher Scientific). Chemiluminescent signal was examined by LAS 4000 biomolecular imager and analyzed using ImageQuant™ software (GE Healthcare Life Sciences).
Las 4000 biomolecular imager
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The LAS 4000 biomolecular imager is a compact and versatile system designed for high-resolution imaging of gels, blots, and other biomolecular samples. It utilizes a charge-coupled device (CCD) camera and advanced optics to capture detailed images, providing researchers with a powerful tool for analysis and documentation of their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent nucleic acid detection kit, by Thermo Fisher Scientific (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Zeta probe gt membrane, by Bio-Rad (1 mentions)
Biotin 3 end dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Ecl system, by Cytiva (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Versamax, by Molecular Devices (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
4 protocols using las 4000 biomolecular imager
1
Biotin-labeled DNA extraction and detection
2
Extraction and Analysis of Cellular Proteins
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Cells were disrupted on ice for 30 min in cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, and 1 mM PMSF [phenylmethylsulfonyl fluoride]) with protease inhibitor cocktail (Roche, Switzerland). The supernatant fraction was harvested after centrifugation at 13,000 rpm for 10 min. Protein concentrations were determined with the Bio-Rad protein assay reagent (Richmond, CA). For nuclear and cytosol fractions, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA) were used according to the manufacturer's protocol. The protein lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes in 20 mM Tris-HCl (pH 8.0), containing 150 mM glycine and 15% (v/v) methanol. Membranes were blocked with 5% non-fat dry milk in 1 × TBS containing 0.05% Tween 20 (TBS-T) and incubated with primary antibodies at 4 °C overnight. Protein bands were visualized by the LAS4000 Biomolecular Imager (GE Healthcare, United Kingdom) and Western Lightning Plus ECL (Perkin Elmer, Waltham, MA) after incubation with appropriate secondary antibodies.
3
Immunoblotting with DJ-1 Detection
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Immunoblotting was performed as previously described29 . Signals were visualized by chemiluminescence (ECL system, Amersham Biosciences, Piscataway, NJ) and detected with an LAS4000 biomolecular imager (GE healthcare). The primary antibody against DJ-1 was obtained from Abcam (UK), and other primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The horseradish peroxidase-conjugated secondary antibodies were purchased from KPL (Gaithersburg, MA). The blots were quantified using NIH ImageJ software and normalized to actin.
4
Protein Expression Analysis of Spinal Cord
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Spinal cord tissues were collected at DPI-1 and DPI-28 and washed with ice-cold phosphate-buffered saline, placed at 4 °C, homogenized in 180 lysis buffer (PRO-PREP™, iNtRON Biotechnology, Seongnam, Korea), and centrifuged at 14,000 rpm at 4 °C for 15 min. The supernatant was collected to determine protein concentration using a Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA). Protein concentration was determined by a VersaMax™ microplate reader (Molecular Devices, San Jose, CA, USA). Equal amounts of protein (40 μg) were separated electrophoretically by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to polyvinylidene fluoride membranes (#162-0177, Bio-Rad, Hercules, CA, USA). The membranes were then incubated for 1 h with 5% non-fat skim milk prepared in TBS buffer to block nonspecific binding. The membranes were then incubated overnight in the cold room with primary antibodies after 1-h incubation at room temperature with corresponding secondary antibodies. The blots were visualized with enhanced chemiluminescence (ECLTM, GE Healthcare, Chicago, IL, USA), using the LAS 4000 biomolecular imager (GE Healthcare, Chicago, IL, USA). The immunoblotting was quantified using ImageJ software (Fiji).
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