Western blot was performed as described previously (11 (link)). Briefly, extracted proteins were separated on 10 or 12% SDS–polyacrylamide gels and electro-transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS), followed by overnight incubation with primary antibody at 4°C. Afterward, the membranes were washed and probed with HRP-conjugated anti-rabbit or anti-mouse IgG. The signals in the membrane were visualized by Chemi-Lumi One L (NacalaiTesque, Kyoto, Japan) and captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan). β-tubulin or β-actin was used as an internal loading control. Quantification of the bands was performed using ImageJ software.
Luminescent image analyzer las 1000
Manufactured by Fujifilm
Sourced in Japan, United States
The Luminescent Image Analyzer LAS-1000 is a compact and versatile imaging system designed for the analysis of luminescent samples. It captures high-quality images of bioluminescent, chemiluminescent, and fluorescent samples with a CCD camera and specialized optical system. The LAS-1000 provides accurate and reproducible data for a variety of applications, including western blotting, gel documentation, and protein expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chemi lumi one l, by Nacalai Tesque (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Enhanced chemiluminescence system, by Nacalai Tesque (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Horseradishperoxidase conjugated anti rabbit or mouse igg antibody, by Cell Signaling Technology (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Acrylamide, by Nacalai Tesque (1 mentions)
O glycosidase, by New England Biolabs (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Mab1859, by R&D Systems (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Nitrocellulose membrane, by Roche (1 mentions)
Total p38, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit igg h l, by Promega (1 mentions)
Signalboost immunoreaction enhancer kit, by Merck Group (1 mentions)
Imagegauge 3, by Fujifilm (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
27 protocols using luminescent image analyzer las 1000
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sample preparation and Western blot analysis were performed as we previously reported [4 (link)]. Briefly, liver and kidney tissues were homogenized in urea buffer (8 mol/L urea, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L Tris-HCl at pH 8.0) on ice. Lysates were sonicated and centrifuged at 12,000 rpm for 30 min at 4 °C. Supernatants were collected and assayed for protein concentration with Pierce Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For cells, the cellular protein was extracted with 1× SDS sample buffer. The same amount of protein was loaded and separated by SDS-polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes, which were followed by blocking the membranes with 5% non-fat dry milk in PBS and incubation with primary antibody overnight at 4 °C. After washing, the membranes were probed with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG, and the bands were developed using Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan) and captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan). The results were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). β-actin or GAPDH was used as internal loading control.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extraction and western blot analysis were carried out as described previously [7 (link),9 (link)]. Briefly, cellular proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking the nonspecific binding with 5% nonfat dry milk or 3% BSA for 1 h, the membranes were reacted with the first antibodies overnight at 4°C, which was followed by incubation with the second horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody. The bands in the membranes were visualized using Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan) and captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan).
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein was extracted by suspending the cells in sodium dodecyl sulphate (SDS) lysis buffer together with freshly added proteinase inhibitor cocktail (Nacalai tesque, Kyoto, Japan). Lysates were incubated on ice for 30 min with intermittent mixing and then centrifuged at 12,000 rpm for 10 min at 4 °C in an Eppendorf centrifuge. The supernatant was recovered and the protein concentration was determined using the Micro BCA Protein Assay Kit (The Thermo Scientific Pierce, Rockford, IL). Western blot was performed using the enhanced chemiluminescence system. Briefly, extracted cellular proteins were separated by 10% SDS polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. After blocking with 3% bovine serum albumin in PBS, the membranes were incubated with primary antibody for 1.5 h at room temperature or at 4 °C overnight. After washing, the membranes were probed with horseradishperoxidase-conjugated anti-rabbit or -mouse IgG antibody (Cell Signaling, Beverly, MA), and the bands were visualized by using the enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan). The chemiluminescent signal was captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan) and quantified with the Image J software (http://rsb.info.nih.gov/ij ). To confirm equal loading of proteins, the membranes were probed for β-actin.
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extraction and western blot analysis were performed as described previously 5 . Briefly, extracted proteins were loaded onto 10% SDS–polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes. After blocking with 5% non‐fat dry milk or 3% BSA in PBS, the membranes were incubated with primary antibody overnight at 4°C. After washing, the membranes were probed with horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG, and the bands were visualized using Chemi‐Lumi One L (Nacalai Tesque, Kyoto, Japan). The chemiluminescent signal was captured with a Fujifilm luminescent image LAS‐1000 analyzer (Tokyo, Japan). GAPDH was used as an internal loading control.
6
Gel Contraction Assay for BSMC Phenotypic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gel contraction assays were performed as described by Yao et al.39 (link). Briefly, 1 × 105/ml BSMCs in Dulbecco’s modified Eagle’s medium were mixed with a collagen solution and incubated in 24-well plates at 37 °C. After gel formation, the cells were exposed to AICAR, FFA or metformin. The images of the gels were captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan). The surface area was measured using ImageJ software.
7
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular proteins were extracted with 1× SDS lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol) that was supplemented with a proteinase inhibitor cocktail or protein kinase inhibitor before use. Protein concentration was measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The same amount of proteins were separated by 10% SDS-PAGE, which was followed by protein transfer to PVDF membranes with wet-blotting apparatus. After treatment of the membrane with 5% skimmed milk or 1% BSA in 0.05% Tween-20 PBS solution (TPBS) for 1 h, the membranes were incubated with primary antibody overnight at 4 °C, followed by washing with TPBS and incubation with peroxidase-conjugated secondary antibody for an additional 1 h. The signal in the membrane was detected using the enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan) and captured with a Fujifilm luminescent image LAS-1000 analyzer (Tokyo, Japan). The intensity of the bands was quantified with the NIH ImageJ software (http://rsb.info.nih.gov/ij , accessed on 1 May 2021). The equal loading of sample protein in each lane was confirmed by probing the blot with β-actin or staining with EZ blue.
8
Dectin-1 Glycosylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2B4 cells expressing hDectin-1A or 1B (2×106 for glycosidase analysis, 1×108 for OpeRATOR treatment) and splenocytes (6×107) isolated from hDectin-1 transgenic mice were lysed with NP-40 lysis buffer. The solubilized hDectin-1 was captured on beads with an anti-hDectin-1 mAb (MAB1859, R&D Systems, Minneapolis, MN). For glycosidase analysis, the immobilized proteins were treated with PNGase F or PNGase F plus O-glycosidase mix including Neuramindase, Hexosaminidase, and O-glycosidase (New England Biolabs, Beverly, MA) according to the manufacturer’s instruction. O-glycosylated peptides were liberated by OpeRATOR and SialEXO (Genovis, Lund, Sweden) according to the manufacturer’s instructions, electrophoresed through 10% acrylamide (Nacalai Tesque) or 10–20% gradient gels (ATTO, Tokyo, Japan) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). PVDF membranes were detected using anti-hDectin-1 polyclonal antibodies (AF1859, R&D Systems), followed by incubation with an HRP-labeled secondary IgG (Invitrogen, Carlsbad, CA) or Protein G (Sigma-Aldrich, St. Louis, MO). Immunocomplexes were detected using Chemi-Lumi One Super (Nacalai Tesque) and a Luminescent Image Analyzer LAS-1000 (Fujifilm, Tokyo, Japan).
9
Western Blot Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Roche Diagnostics, Rotkreuz, Switzerland). These nitrocellulose membranes were blocked with 5% skimmed milk powder/Tris-buffered saline with 0.1% Tween20 (w/v) at 4 °C overnight. After that, the membranes were incubated with the primary antibodies:inhibitor of κB kinase-α (IκB-α; Santa Cruz Biotechnology, Delaware Ave Santa Cruz, CA, USA; dilution ratio; 1:1000), TLR4, CD14 (Santa Cruz Biotechnology, USA; dilution ratio; 1:200), p-p38, p-JNK, p-ERK, total p38, total JNK, total ERK (Cell Signaling Technology, Boston, CA, USA; all at 1:200), and β-actin (Cell Signaling Technology, Boston, CA, USA; 1:500); the samples were incubated overnight at 4 °C. Then, membranes were treated with HRP-conjugated anti-rabbit IgG (H + L) as the second antibody (Promega, Madison, WI, USA; dilution ratio; 1:2000). Chemiluminescent HRP Substrate examined the immunoblotting (Cat. NO: WBKLS0100; ImmobilonTMWestern, MA, USA) according to the manufacturer’s instructions. Membranes were exposed by a FUJIFILM Luminescent Image Analyzer LAS-1000 (Macintosh TM, USA). The intensities of the resulting bands were quantified by Quantity One software on an AGS-800 densitometer (BioRad, Hercules, CA, USA).
10
Quantification of CYP2D Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CYP2D protein levels in the brain and liver microsomes of control and lurasidone-treated rats were estimated by Western immunoblot analysis, as described previously [15 (link)]. The total protein concentration in samples was determined in the microsomes using the Lowry methods [26 (link)]. All samples were heated in a Laemmli sample buffer for 5 min at 100 °C. Next, the microsomal proteins (10 μg) were separated using an SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The blots were probed with the following primary antibodies: rabbit antirat CYP2D4 (1:1000) or rabbit antihuman CYP2D6 (1:2000). In order to dilute the primary and secondary antibodies, the specified solutions from the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore, Burlington, MA, USA) were used. As a positive control, we used rat cDNA-expressed CYP2D4 (2.5 µg) or human CYP2D6 (1 µg). The blots were visualized using the Luminescent Image Analyzer LAS-1000 and Image Gauge 3.11 programs (Fuji Film, Tokyo, Japan). The results were normalized to β-actin protein level.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!