Nuclease stability of DNA NPs was investigated following their incubation for 30 min at 37 °C in the presence of different concentrations of DNase I. DNase I buffer was made-up at 0.025 U/μL, 0.0025 U/μL and 0.00025 U/μL as described in the manufacturers guide (RNase-Free DNase, Qiagen). Following DNase treatment, complexes and naked pDNA were treated with proteinase K (20 μg) to remove peptide and allow visualisation of DNA by a gel sift assay. Samples were run on a 1% agarose gel (with 0.5 μg/mL ethidium bromide (EtBr)) in 1× trisacetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer and visualized on a Luminescent Image Analyser LAS-4000 (FUJIFILM).
Luminescent image analyser las 4000
Manufactured by Fujifilm
Sourced in United States, Japan
The Luminescent Image Analyser LAS-4000 is a piece of lab equipment designed for the analysis of luminescent samples. It provides high-sensitivity detection and quantification of various luminescent signals, such as chemiluminescence and bioluminescence, without the need for radioactive labeling.
Automatically generated - may contain errors
Lab products found in correlation
Rnase free dnase, by Qiagen (2 mentions)
Las 4000, by Cytiva (2 mentions)
Amicon ultra 0.5 ml, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Low range ssrna ladder, by New England Biolabs (1 mentions)
Amicon ultra 4 ml centrifugal filter unit, by Merck Group (1 mentions)
Sybr green 2, by Lonza (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Cytiva (1 mentions)
Anti ha hrp, by Roche (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Nonfat dried milk powder, by AppliChem (1 mentions)
Anti β actin, by MP Biomedicals (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Sc 81178, by Santa Cruz Biotechnology (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Ecl prime western blotting detection system, by GE Healthcare (1 mentions)
8 protocols using luminescent image analyser las 4000
1
Nuclease Stability of DNA Nanoparticles
2
Cupric Sulfate Charring of Biomolecules
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After development, plates were sprayed uniformly with 10% cupric sulphate in 8% aqueous phosphoric acid [11 (link),49 (link)], allowed to dry for 10 min at room temperature and then heated to 145 °C for 10 min. Imaging of cupric-sulphate charred plates was carried out using the Luminescent Image Analyser LAS-4000 (Fuji Film, Tokyo, Japan). All images were analysed using Multi Gauge V3.0 software.
3
Denaturing PAGE for RNA Purification
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Enzymatic reactions were analysed by denaturing PAGE [4–8% polyacrylamide (acrylamide/N, N′-methylenebisacrylamide, 19:1), 7.5 M urea, 25% (v/v) formamide, 89 mM Tris, 89 mM boric acid, 2 mM EDTA]. The RNA samples were mixed with equal amount of 2× formamide loading solution [80% (v/v) formamide, 10 mM EDTA pH 8.0, 0.1 mg/mL xylene cyanol FF, 0.1 mg/mL bromophenol blue], and heated at 90 °C for 3 min before being loaded on the gel. The gels were electrophoresed and stained with SYBR Green II (Lonza) and visualised on a BioRad ChemiDoc XRS+ System (BioRad, Hercules, CA, USA), Luminescent Image Analyser LAS 4000 (Fujifilm, Tokyo, Japan) or FAS-IV Imaging System (Nippon Genetics, Tokyo, Japan). Low Range ssRNA Ladder (New England Biolabs, Ipswich, MA, USA) was used as the size marker. RNAs were also purified by preparative denaturing PAGE. The RNA bands were visualised by UV shadowing, and crushed and extracted with water. The extracts were desalted by centrifugation with Amicon Ultra-0.5 mL or Amicon Ultra-4 mL centrifugal filter unit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. The RNAs were then precipitated with sodium acetate (pH 5.2) and 2-propanol.
4
Western Blot Analysis of Notch Ligands
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Cells/embryos were lysed in 2x sample buffer (0.125 M Tris pH 6.8/4% SDS/20% glycin/5% beta-mercaptoethanol/0.025% bromphenol blue). Proteins were separated by SDS-PAGE and transferred to Immobilon-P Transfer membranes (Millipore) by wet tank blotting. Blots were blocked in 5% nonfat dried milk powder (AppliChem) in PBS/0.1% Tween 20. Primary antibodies: anti-HA HRP (rat monoclonal; clone 3F10, Roche; HRP, horseradish peroxidase-conjugated; 1:5,000–1:10,000), anti-Flag HRP (mouse monoclonal; clone M2, Sigma), anti-GFP HRP (mouse monoclonal; MACS molecular; 1:10,000), anti-DLL1 (1F9, rat monoclonal, [21 (link)] 1:1,000), anti-DLL4 (rabbit polyclonal against peptide C-GKIWRTDEQNDTLT; BioGenes; 1:50–1:100), anti-β-actin (mouse monoclonal; MP Biomedicals; 1:250,000–1:500,000), anti-β-tubulin I (Sigma; 1:500,000). Secondary antibodies: anti-mouse HRP, anti-rat HRP, anti-rabbit HRP (Amersham; 1:10,000). HRP was detected with ECL Western Blotting Detection Reagents (Amersham) with the Luminescent Image Analyser LAS-4000 (Fujifilm); signals were quantitated with ImageJ software.
5
Nuclease Stability of DNA Nanoparticles
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Nuclease stability of DNA NPs was investigated following their incubation for 30 min at 37 °C in the presence of different concentrations of DNase I. DNase I buffer was made-up at 0.025 U/μL, 0.0025 U/μL and 0.00025 U/μL as described in the manufacturers guide (RNase-Free DNase, Qiagen). Following DNase treatment, complexes and naked pDNA were treated with proteinase K (20 μg) to remove peptide and allow visualisation of DNA by a gel sift assay. Samples were run on a 1% agarose gel (with 0.5 μg/mL ethidium bromide (EtBr)) in 1× trisacetate-ethylenediaminetetraacetic acid (EDTA) (TAE) buffer and visualized on a Luminescent Image Analyser LAS-4000 (FUJIFILM).
6
Jurkat Cell Protein Extraction and Western Blot
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The protein of Jurkat cells was extracted using a PRO-PREP (Intron Biotechnology, Seoul, Republic of Korea). The protein concentration was determined using Bio-Rad protein assay reagent according to the manufacturer’s instruction and BSA (Bio-Rad, Hercules, CA, USA) was used as a standard for quantification. Equal protein amounts were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were incubated for 1 h with blocking solution (5% skim milk) at room temperature and followed by incubation for primary antibodies overnight at 4 °C. p-Lck (Y394) antibody (1:500, MBS128234, MyBioSource) was used as a primary antibody, and β-actin (1:1000, sc-81178, Santa Cruz Biotechnology) was used as an internal control. And then membranes were incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The membranes were analysed using an enhanced chemiluminescence (ECL) substrate and imaged by LAS-4000 luminescent image analyser (FUJIFILM, Tokyo, Japan).
7
Western Blot Protein Detection
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Proteins transferred to Immun-Blot PVDF Membranes (Bio-Rad) were reacted with anti-fibrinogen β-chain, anti-plasminogen, and anti-αSMA primary antibodies. The proteins were visualised using an ECL Prime Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Images were taken using an LAS-4000 luminescent image analyser (Fujifilm Life Science Stamford, CT, USA).
8
Western Blot Analysis of Apoptotic Cells
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THP1 or epHero THP1 cells were rendered apoptotic as described above, pelleted, and resuspended in cell lysis buffer containing protease and phosphatase inhibitors. Samples were sonicated on ice, and protein concentration determined via a BCA assay. Following this, 10 µg protein was loaded per well and electrophoresed through 4–12% SDS-PAGE gels before being transferred to a PVDF membrane as previously described [41 (link)]. The membranes were blocked in a solution comprising Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) skim milk and incubated overnight at 4 °C with anti-mCherry (1:2 000) and anti-eGFP (1:1 000) antibodies. Incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10 000) was performed at room temperature for 1 h. To detect β-actin, membranes were incubated with an HRP-conjugated anti-β-actin antibody (1:10 000) for 1 h at room temperature. Membranes were developed on the LAS4000 Luminescent Image Analyser (Fujifilm Life Science, Cambridge, Massachusetts, USA) using the West Pico or West Femto ECL systems.
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