Cells were lysed in sample buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl pH 6.8), and the conditioned media were collected for methanol precipitation. After protein separation by SDS-PAGE, Western blotting was conducted using an anti-Flag M2 mouse monoclonal antibody (1:10,000, Sigma-Aldrich, Saint Louis, MO, USA). A chemiluminescent substrate was used for the detection of HRP-coupled secondary antibodies using a LAS4000 detection system (GE Healthcare, Chicago, IL, USA).
Las 4000 detection system
Manufactured by GE Healthcare
Sourced in United States
The LAS-4000 is a detection system designed for high-performance imaging of chemiluminescent and fluorescent signals. It features a highly sensitive CCD camera, a motorized lens, and advanced software for image capture and analysis. The core function of the LAS-4000 is to provide researchers with a reliable and efficient tool for detecting and quantifying a wide range of biological and chemical signals.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (4 mentions)
Hybond n membrane, by GE Healthcare (3 mentions)
Cdp star reagent, by Roche (3 mentions)
Northernmax gly sample loading dye, by Thermo Fisher Scientific (3 mentions)
Dig northern blot starter kit, by Roche (3 mentions)
Semi dry blotting system, by Bio-Rad (3 mentions)
Hybond, by Cytiva (3 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Lumi light, by Roche (1 mentions)
Gapdh, by GeneTex (1 mentions)
5 protocols using las 4000 detection system
1
Protein Expression and Detection
2
Quantification of Circular RNAs in Primate Brain
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Total RNA (10 μg for 10- and 20-year-old rhesus macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1 × TBE for 20 min and transferred to a Hybond-N + membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/ cm2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using DIG Northern Blot Starter KIT (Roche) DIG-labeled in vitro transcribed circCACNA2D1, circCACNA1E, CDRA1s, and circMbl junction site-targeting probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare).
3
Northern Blot Analysis of Gria1 in Macaque Brain
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Total RNA (10 μg for 10- and 20-year old male macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1×TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/cm2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using the DIG Northern Blot Starter Kit (12039672910, Roche). DIG-labeled in vitro transcribed Gria1 junction site-targeting circular, and junction site-nontargeting linear probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare). The primer sequences of probes were seen in Supplementary Data 3 .
4
Protein Extraction and Western Blotting Protocol
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Fresh frozen tissues were lysed using the TissueLyser (Qiagen,) and 4x SDS-lysis buffer (250mM Tris/HCl, 34% (w/v) Glycerin, 8.2% (w/v) SDS, 5% (v/v) Δ-Mercaptoethanol, pH 6.8) supplemented with protease inhibitor cocktail (Roche). Total cell extracts were generated by scraping cells from the cell culture plate applying a 4x SDS-lysis buffer. After sonication, protein concentration of samples was measured by OD280. Equal amounts were loaded onto SDS-PAGE gels [29 (link)] and electrotransferred onto nitrocellulose membrane (0.1 μm, GE Healthcare). Immunostaining was performed with primary antibody followed by detection with horseradish-peroxidase conjugated antibodies (Pierce). A Polyclonal antibody against CXorf61 (anti-CXorf61-B) was purchased from Sigma-Aldrich (HPA004773). Actin antibody was from Sigma-Aldrich (A1978), Histon H3 antibody (D1H12) from Cell Signalling (4499L), Lamin B from Progen (61047C) and GAPDH from GeneTex (GTX627408). Chemiluminescence analysis was done using lumi-light (Roche), dura or femto reagent (Pierce) in the LAS 4000 detection system (GE Healthcare).
5
Northern Blotting of lncMtDLoop in AD
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Total RNA (10 μg for human postmortem PFC and hippocampal frozen tissues of AD and control individuals as well as wild type and 3xTg mouse brain fresh tissues, 2 μg for mouse primary neurons) was denatured using NorthernMax ® -Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1×TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UVcrosslinked with 150 mJ/ cm 2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using DIG Northern Blot Starter KIT (12039672910, Roche). DIG-labeled in vitro transcribed lncMtDLoop, and control probes were hybridized overnight. The membranes were washed three times in 2× SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2× SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare). The primer sequences of probes were seen in the Supplementary Table 2.
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