To identify the expressed his-tag fusion proteins in the different elution fractions of the Ni-NTA agarose affinity chromatography, aliquots of the elution fractions were analyzed by SDS-PAGE and the separated protein bands were transferred to a nitrocellulose membrane (Thermo Fisher Scientific Inc., Waltham, MA, USA) by a wet blotting method. The blots were reversibly stained with Ponceau red (Sigma-Aldrich, St. Louis, MO, USA) to ensure complete protein transfer. Subsequently the blotting membrane was blocked using the blotting-grade blocker solution (Bio-Rad, Hercules, CA, USA) and was then incubated for 1 h with a 1:5000 diluted anti-his-tag antibody carrying horse redish peroxidase as marker enzyme (Miltenyi Biotec GmbH, Bergisch Gladbach, Deutschland). Immunoreactive bands were visualized using the SERVALight Polaris CL HRP WB Substrate Kit (Serva Electrophoresis GmbH, Heidelberg, Germany). Chemiluminescence was detected using a FUJIFILM Luminescent Image Analyzer LAS-1000plus & Intelligent Dark Box II.
Intelligent dark box 2
Manufactured by Fujifilm
Sourced in Japan
The Intelligent Dark Box II is a photographic equipment designed to provide a controlled and consistent environment for processing photographic materials. It is a self-contained, automated system that regulates temperature, humidity, and exposure to light, ensuring optimal conditions for film development and other photographic processes.
Automatically generated - may contain errors
Lab products found in correlation
Servalight polaris cl hrp wb substrate kit, by Serva Electrophoresis (3 mentions)
Luminescent image analyzer las 1000plus, by Fujifilm (3 mentions)
Protean 2 xi cell electrophoresis system, by Bio-Rad (2 mentions)
Las 1000 scanner, by Fujifilm (2 mentions)
Protran ba 85 membrane, by Carl Roth (2 mentions)
Ponceau red, by Merck Group (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Blotting grade blocker solution, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Trizma base, by Merck Group (1 mentions)
Las 1000 camera, by Fujifilm (1 mentions)
Ecl select, by GE Healthcare (1 mentions)
Las 1000, by Fujifilm (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Fluor s max multiimager, by Bio-Rad (1 mentions)
Prism program, by GraphPad (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Western lightning plus ecl enhanced chemiluminescence substrate, by PerkinElmer (1 mentions)
Amersham ecl western blotting, by Cytiva (1 mentions)
Supersignal west dura ecl substrate, by Thermo Fisher Scientific (1 mentions)
14 protocols using intelligent dark box 2
1
Detecting His-Tag Fusion Proteins in Chromatography Fractions
2
Myc-tag Protein Detection by Western Blot
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3 OD600 units of cells were harvested by centrifugation, resuspended in 100 μl of 1.85 M NaOH containing 7% β-Mercaptoethanol and incubated on ice for 10 min, followed by addition of 100 μl of 50% TCA and incubation on ice for 5 min. After centrifugation at 13,000 g for 10 min, protein pellets were washed twice with 1 M Trizma base (Merck, Darmstadt, Germany) before resuspension in 100 μl 2× SDS-PAGE loading buffer (100 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 4 mM EDTA, 0.2% bromophenol blue, 2% β-Mercaptoethanol). Samples were incubated at 95°C for 10 min, briefly centrifuged and analyzed by SDS-PAGE and immunoblotting using anti-Myc antisera (9E11, Santa Cruz Biotechnology, Dallas, Texas, USA). Immunoreactive signals were detected with a horseradish peroxidase-conjugated secondary antibody (P0447, Dako, Santa Clara, California, USA) reacted with ECL Select (GE Healthcare, Little Chalfont Buckinghamshire, United Kingdom) and images were acquired with a FujiFilm LAS-1000 camera using the Intelligent Dark Box II (FujiFilm, Tokyo, Japan).
3
Quantitative Western Blot Analysis
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Western blot was performed as previously described.[32 (link)] The antibodies and reagents used are described in the next section. Imaging was done on a Fuji Intelligent Dark Box II with LAS-1000 software [Fuji, Tokyo, Japan] and Bio-rad FluorS MAX MultiImager with Bio-Rad Quantity One software [Bio-Rad Laboratories Inc., Hercules, CA, USA]. Images were analysed with ImageJ software [National Institutes of Health, Bethesda, MD, USA]. Intensities of protein-specific bands were calculated relative to respective Actin intensity to account for loading irregularities.[32 (link)]
4
Western Blot Analysis of OmpP5 Protein
5
SDS-PAGE Analysis of Bacterial Lysates
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For SDS/PAGE, approximately 100 µg denatured protein of the bacterial lysate supernatants were analyzed on a 7.5% polyacrylamide gel. The separated protein bands were then transferred onto a Protran BA 85 Membrane (Carl Roth GmbH, Karlsruhe, Germany) and the blots were probed with an anti-his-tag-HRP conjugated antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Immunoreactive bands were visualized using the SERVALight Polaris CL HRP WB Substrate Kit (Serva Electrophoresis GmbH, Heidelberg, Germany). Chemiluminescence was detected on a FUJIFILM Luminescent Image Analyzer LAS-1000plus & Intelligent Dark Box II. For testing the pH-dependence of the product mixture of wildtype human and mouse ALOX15B orthologs, a 1:1 mixtures of 0.05 M sodium phosphate buffer and 0.05 M sodium borate buffer were used and the different pH values were adjusted at room temperature by the addition of 5 M NaOH or HCl, respectively. The protein concentrations in the bacterial lysates were quantified using Bradford Reagent for quantitative protein determination (AppliChem, VWR International GmbH, Darmstadt, Germany) according to the instructions of the vendor. For statistical calculations and figure design we used the GraphPad prism program (version 8.00, GraphPad Software, La Jolla, CA, USA).
6
Western Blotting Protocol for Protein Detection
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Proteins in cell lysates were separated in 12.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and probed with the antibodies described above and developed using Amersham ECL Western blotting detection reagent (GE Healthcare) and X-ray film (Fig. 1G and 3C ) or using Western Lightning Plus-ECL enhanced-chemiluminescence substrate (PerkinElmer), and proteins were detected under an Intelligent dark box II (Fujifilm) (Fig. 2B ). As controls for loading and transfer, the blots were probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or anti-β-actin.
7
Western Blotting Procedure for Protein Detection
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Equal volumes of cytoplasmic and nuclear extracts (adjusted by blotting with cytoplasmic and nuclear markers) were boiled in SDS sample buffer containing 10 mM DTT and separated by SDS-PAGE. Proteins were electrotransferred to polyvinylidene difluoride membrane (Immobilon P) and blocked for 1 h in 5% BSA in PBS containing 0.04% Tween 20. Membranes were incubated with primary antibody, diluted in 1% BSA in PBS at 4°C overnight, washed three times in PBS, and incubated with horseradish peroxidase–conjugated anti–rabbit (1:40,000 dilution) or anti–mouse (1:25,000 dilution) antibody (Amersham Pharmacia). Proteins were visualized using Super Signal West Dura ECL substrate (ThermoScientific), and membranes were scanned with a charge-coupled device camera Intelligent Dark Box II (Fujifilm). Raw data files were exported as TIFF files from Aida software, and level autocontrast was applied to the blots using Photoshop (Adobe).
8
SARS-CoV-2 Protein Expression and Detection
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Cells were harvested, lysed with lysis buffer (20 mM Tris-HCl; pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) supplemented with cOmplete protease inhibitor cocktail (11697498001, Roche). The lysates were electrophoresed on 8-20% SDS-polyacrylamide gels. Then samples were transferred to polyvinylidene difluoride membranes (Millipore), and blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 (TBS-Tween). Primary antibodies were as follows: anti-SARS-CoV-2-S antibody (GTX632604, GeneTex, 1/1000 in 5% milk TBS-Tween), anti-SARS-CoV-1-M antibody (AP6008b, Abgent, 1/1000 in 5% milk TBS-Tween), anti-SARS-CoV-2-N antibody (GTX135357, GeneTex, 1/1000 in 5% milk TBS-Tween), anti-SARS-CoV-2-ORF3a antibody (A20234, ABclonal, 1/1000 in 5% milk TBS-Tween), anti-SARS-CoV-2-NSP6 antibody (9177, ProSci, 1/1000 in 5% milk TBS-Tween), and anti-tubulin (T5168, Sigma-Aldrich, 1/10,000 in 5% milk TBS-Tween). Secondary antibodies were as follows: anti-mouse IgG-HRP (330, MBL, 1/10,000 in 5% milk TBS-Tween), anti-rabbit IgG (458, MBL, 1/10,000 in 5% milk). The signals were detected through the enhanced chemiluminescent substrate (WBKLS0100, Millipore, or 34094, Thermo Fisher Scientific) by Intelligent Dark BoxII (Fujifilm) using IR LAS-1000 Pro (version 2.5) software. Uncropped scans of the most important western blots are provided in the Source Data file.
9
Immunoblot Analysis of Cell Lines
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CAFs (2 × 104 cells per 12-well dish), Hep3b and SNU423 (5 × 105 cells per 60 mm dish) and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 105 cells per 60 mm dish), after the specified treatments, were washed in ice-cold phosphate buffer saline (PBS), pH 7.4, lysed and analyzed by immunoblot as described20 (link), with antibodies at the following dilutions: fibronectin, 1:10,000 (Sigma-Aldrich, Stockholm, Sweden, F3648); fatty acid synthase (FASN), 1:1,000 (ab22759); calponin, 1:2,000 (EP798Y, ab46794); LXRα, 1:1,000 (ab41902); Smad3, 1:1,500 (ab40854), all from Abcam, Cambridge, United Kingdom; α-smooth muscle actin (αSMA), 1:500 (Santa Cruz Biotech Inc., Santa Cruz, CA, USA, sc1a4); GAPDH, 1:50,000 (Ambion, ThermoFisher Scientific, Fyrislund, Sweden, AM4300). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (ThermoFisher Scientific, Fyrislund, Sweden) were used at 1:20,000 dilution. Triplicate (nb = 3) biological experiments were performed in 2 technical replicates (nt = 2) per condition. Densitometric quantification of protein bands was performed using the Fujifilm Intelligent Dark Box II program of a Fuji Aida digital scanner (Fujifilm Nordic AB, Stockholm, Sweden).
10
Luminol-Based Chemiluminescent Imaging
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Chemiluminescent assays were performed in the presence of luminol 220 µM and glucose 100 mM in a pH 8.5 buffered solution composed of Veronal at a concentration of 30 mM and KCl at a concentration of 30 mM (VBS). Fanciful balls were immersed in VBS containing both substrates and the chemiluminescent signal imaged using a charged coupled device (CCD) cooled camera (Las-1000 Plus, Intelligent Dark Box II, FUJIFILM, Tokyo, Japan). Integration times are indicated in minutes on result pictures.
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