HEK293T cells were seeded in a 12-well plate at a density of 2 × 105 cells/well and cultured for 24 h. Next, the cells were transiently transfected with each spike plasmid and cultured for two days. Thereafter, the cells were lysed using ULTRARIPA A buffer (BioDynamics Laboratory Inc., Tokyo, Japan). Cell lysates were treated with PAA at the indicated concentration for 10 min. Then, the amount of protein in the cell lysates was measured using BCA protein assay kit (Takara Bio Inc., Shiga, Japan). Equal amount of protein was determined by SDS-PAGE and electrotransferred to an Immobilon-P PVDF membrane (EMD Millipore, Billerica, MA, USA). Western blotting was performed using the standard method, and the following antibodies were used: anti-SARS-CoV-2 spike (1A9, GTX632604, mouse monoclonal antibody, GeneTex, Irvine, CA, USA), anti-SARS-CoV-2 nucleocapsid (HL344, GTX635679, rabbit monoclonal antibody, GeneTex), anti-GAPDH (3H12, M171-3, mouse monoclonal antibody, MBL), and horseradish peroxidase-conjugated sheep anti-mouse IgG (NA931, Amersham Biosciences, Amersham, UK). Immunoblot signals were developed using EzWestLumi plus (ATTO Corp., Tokyo, Japan) and recorded with an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Tokyo, Japan).
Ezwestlumi plus
Manufactured by ATTO Corporation
Sourced in Japan
The EzWestLumi plus is a lab equipment product developed by ATTO Corporation. It is designed for visualization and analysis of proteins and nucleic acids in gel electrophoresis experiments. The core function of this product is to capture and analyze chemiluminescent and fluorescent signals generated during Western blotting and other gel-based assays.
Automatically generated - may contain errors
Lab products found in correlation
C digit blot scanner, by LI COR (5 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg h l antibody, by Bio-Rad (2 mentions)
Luminograph 2, by ATTO Corporation (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Imagequant las4000 mini image analyzer, by GE Healthcare (1 mentions)
Hl344 gtx635679, by GeneTex (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Anti gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Lysopure nuclear and cytoplasmic extractor kit, by Fujifilm (1 mentions)
Cleaved caspase 3 antibody 9661, by Cell Signaling Technology (1 mentions)
Anti clu sc 5289, by Santa Cruz Biotechnology (1 mentions)
Ez capture mg, by ATTO Corporation (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Anti rabbit igg hrp, by Cytiva (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
24 protocols using ezwestlumi plus
1
SARS-CoV-2 Spike Protein Analysis
2
Quantitative Analysis of Serum Amyloid A4
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Plasma was diluted 1 : 100 with electrophoresis sample buffer and 10 μL of it was subjected to tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transfer to a polyvinyl difluoride membrane, as previously described [7 ]. The membrane was blocked in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 hour at room temperature (RT) and then reacted with 2 μg/mL rabbit anti-human SAA4 antibodies [9 (link)] for 1 hour at RT, followed by reaction with 1 : 1000-diluted peroxidase-conjugated anti-rabbit immunoglobulins (Bioscience Inc., USA) for 1 hour at RT. Then, as a substrate, EzWestLumi plus (ATTO Corp., Japan) was applied and the output was analyzed using a chemiluminescent image analyzer system, Ez-Capture MG (ATTO). Using CS Analyzer 3.0 software in that system, the intensities of the two SAA4-corresponding bands were read. The SAA4 concentration of each sample was calculated from the intensities of the two bands by comparison with those of the serum, the SAA4 concentration of which was known [9 (link)].
3
Western Blot Analysis of sCLU
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For detection of sCLU, cytoplasmic components were isolated using a LysoPure Nuclear and Cytoplasmic Extractor kit (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and nuclear components were discarded. Cytoplasmic lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and the proteins were transferred onto polyvinylidene fluoride membranes. Blots were incubated with respective primary antibodies overnight at 4°C, followed by incubation with corresponding secondary antibodies for 1 hour. Proteins were visualized by enhanced chemiluminescence using EzWestLumi Plus and Ez‐Capture MG (ATTO Corp., Tokyo, Japan). Anti‐CLU (sc‐5289) and anti‐GAPDH (sc‐32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti‐p‐Akt (4060), anti‐pan Akt (2920), anti‐p‐Erk (1/2) (9106), and anti–phosphorylated mammalian target of rapamycin (p‐mTOR) (5536) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti‐Erk (1/2) antibody (MAB1576) was purchased from R&D Systems (Minneapolis, MN). Cleaved Caspase‐3 antibody (#9661) was purchased from Cell Signaling Technology (Danvers, MA).
4
Immunoblotting of SARS-CoV-2 Viral Proteins
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Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Cat#16488-34, Nacalai tesque, Inc., Kyoto, Japan)-containing protease inhibitors. Proteins were resolved by electrophoresing on a 5–20% gradient sodium dodecyl sulfate polyacrylamide gel and electrotransferred to an Immobilon-P membrane (Cat#IPVH00010, EMD Millipore; Billerica, MA, USA). The primary antibodies against SARS-CoV-2 anti-spike (Cat#GTX632604, clone 1A9), anti-nucleocapsid (Cat#GTX635679, HL344), and anti-membrane (Cat#GTX636245, clone HL1087) proteins were purchased from GeneTex Inc. (Irvine, CA, USA). Cleaved SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP (Cat#7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-rabbit IgG–HRP (Cat#NA934) was purchased from Cytiva™ (Tokyo, Japan). Anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; Cat#171-3, clone 3H12) was purchased from Medical & Biological Laboratories Co., Ltd. (Aichi, Japan). Immunoblot signals were developed using EzWestLumi plus® (Cat#WES-7120, ATTO Corp.; Tokyo, Japan) and recorded with an ImageQuant LAS4000 mini-image analyzer (GE Healthcare Japan Corp.; Tokyo, Japan).
5
Immunoblotting of Protein Samples
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Proteins were resolved by SDS‐PAGE and electrotransferred to the Immobilon‐P membrane (EMD Millipore). Immunoblot was carried out as described previously.13 The following primary antibodies were used: anti‐FLAG M2 (Sigma‐Aldrich), anti‐PD‐L1 (28‐‐8; Abcam), anti‐Sp3 (H‐225; Santa Cruz Biotechnology), anti‐Sp1 (D4C3; Cell Signaling Technology, Inc.), and anti‐glyceraldehyde 3‐phosphate dehydrogenase (anti‐GAPDH, 6C5; Millipore Corp., Chemicon). Horseradish peroxidase–conjugated donkey anti‐rabbit IgG and horseradish peroxidase–conjugated sheep anti‐mouse IgG (both from Amersham Biosciences Corp.) were used as secondary antibodies. Immunoblot signals were developed using EzWestLumi plus® (ATTO Corp.) and recorded with an ImageQuant LAS4000 mini image analyzer (GE Healthcare Japan Corp.). Quantitative analyses of signals were performed using accompanying software ImageGauge ver 4.0.
6
Immunoblot Detection of Methanol Oxidation Enzymes
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Anti-XoxF and anti-MxaF antisera were generated against synthesized peptides corresponding to residues 339–351 of MxaF and residues 374–387 of XoxF1, respectively. Whole-cell lysates of M. trichosporium OB3b and OB3b ΔmxaF mutant were separated on 10% (w/v) acrylamide gel and transferred to a PVDF membrane following general protocols. The Precision Plus Protein™ Dual Color Standards (Bio-Rad Laboratories, Hercules, CA, United States) were used for the molecular weight marker. The blotted membrane was blocked with 5% (w/v) skim milk at room temperature for 1 h and treated at room temperature for 1 h with anti-MxaF or anti-XoxF1 antisera at 1:2,000 dilution in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 (TBS-T; Calbiochem, San Diego, CA, United States). Membrane-bound MxaF and XoxF1 were detected with horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Little Chalfont, United Kingdom) at 1:10,000 dilution in TBS-T and visualized using EzWestLumi Plus (ATTO Corp., Tokyo, Japan) according to the manufacturer’s instructions.
7
Fibroblast Transfection and Protein Expression
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Fibroblasts at a density of 2×105 cells per well were transfected with Ad-FST for 24 hours and lysed in RIPA lysis buffer (ATTO Corp., Tokyo, Japan) containing protease inhibitor (Pierce Mini Tablets, IL, USA). Meanwhile, the culture medium was collected to measure FST protein expression by Ad-FST. Lysates and culture medium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd., Darmstadt, Germany) using a transfer system (Mini Trans-Blot Cell and systems, Bio-Rad, Hercules, CA, USA). The blots were incubated with specific antibodies against MMP-1, MMP-13, TIMP-1, TIMP-2, TIMP-4, fibronectin, PAI-1, TRPV4, and α-SMA (Abcam, Cambridge, UK). After reacting with secondary antibodies, immunoreactive bands were visualized by a Western blot detection system (EzWest-Lumi Plus, ATTO Corp., Japan). To verify the amounts of loaded proteins, blots were stripped of bound antibodies and reprobed using antibodies to actin (Abcam).
8
Quantification of Claudin-4 in Mouse Lung
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Protein extracts of mouse lung tissue were collected as previously described.25 (link) Protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 5% bovine serum albumin (BSA) in 0.1% Tween 20 in Tris-buffered saline (TBS) (21℃, 2 hours) and incubated with anti- claudin-4 (1:200, Abcam Inc., Cambridge, MA, USA) (4℃, overnight), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Detection was performed using EzWestLumi plus (ATTO Corp, Tokyo, Japan). The relative abundance of protein was determined by quantitative densitometry, and the data were normalized to β-actin (Sigma-Aldrich, St. Louis, MO, USA).
9
Western Blot Analysis of Liver Cell Proteins
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The two liver cells (CHANG and HuH-7 cells) were scrapped in RIPA buffer (ab156034), after which the cell were centrifuged at 13,000 rpm, 4ºC for 30 min and supernatant were transfer in new tube for further study. The proteins (20 μg) were separated on TGX stain free gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio Rad, Laboratories Inc., Berkeley, CA, USA). The membrane was then incubated with mouse monoclonal antibody against β-actin (1:12,000 dilutions, Abcam, Cambridge, UK), Bax (1:1000 dilutions, Antibodies-online), Bcl-2 (1:500 dilutions, Santa Cruz) overnight at 4°C. The horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) antibody (1:2000 dilutions Bio Rad) was used as a secondary antibody. Immuno-reactive bands were detected using an EZ west Lumi plus (ATTO corporation, Tokyo, Japan), which is a chemiluminescent substrate to detect HRP on Western blotting membrane. The luminescence intensity (optical density) of the target protein bands was quantified using Lumino Graph 2 (ATTO Corporation). All protein expression levels were normalized to the levels of β-actin protein expression in each band.
10
Striatal Protein Analysis by Western Blot
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Western blot analysis was described in a previous study [36 (link)]. Thirty minutes after the last L-DOPA injection, mice were sacrificed, and the striatum was quickly removed and homogenised in a RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche). Protein samples were separated by 10% SDS-PAGE, and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). Blots were incubated with primary antibodies (Phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, RRID:AB_331646), ERK1/2 (Cell Signaling Technology, RRID:AB_10695746), FosB/ΔFosB (Cell Signaling Technology, RRID:AB_2106903) or actin (Millipore) followed by secondary antibodies, and specific signals were visualised using an enhanced Ez West Lumi plus (WSE-7120L, ATTO Corporation, Tokyo, Japan). Western blot images were quantified using Quantity One 1-D analysis software, version 4.6.1 (Bio-Rad).
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