Western blotting was performed to evaluate the effects of D-allose on the expression in BC cells. After 24 h of incubation, the cells (RT112, 253J, and J82) were treated with varying concentrations of D-allose (10, 25, and 50 mM) for 48 h. Both attached and floating cells were harvested and lysed. Samples were run on a 10% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories Inc. Hercules, CA, USA), transferred onto polyvinylidene difluoride membranes, and blocked in a SuperBlock Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Anti-TXNIP (1:1000) or β-actin (1:1000) were used as the primary antibodies to react with the iBind Flex Western System (Thermo Fisher Scientific). Signals were developed using an ECL (enhanced chemiluminescence) kit (Amersham Biosciences, Buckinghamshire, UK).
Ibind flex western system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBind Flex Western System is a laboratory instrument designed for Western blotting analysis. It provides a platform for efficient protein transfer and immunodetection, enabling researchers to investigate protein expression and modifications.
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Lab products found in correlation
Molecular imager chemidoc xrs, by Bio-Rad (2 mentions)
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Enhanced chemiluminescence ecl kit, by Cytiva (1 mentions)
Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh pab, by Santa Cruz Biotechnology (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Bca kit, by Solarbio (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
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Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Quick coomassie, by Generon (1 mentions)
Nupage 4 12 bis tris midi protein gels, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Sigmafast bcip nbt, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
11 protocols using ibind flex western system
1
D-allose Modulates TXNIP Expression in BC
2
Evaluating SINV Nonstructural Protein Expression
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To determine whether or not altering the capping efficiency impacted the expression of the SINV nonstructural genes, the expression of nsP2 was assessed via Western blotting. Briefly, whole-cell lysates were generated from BHK-21 cells that were infected with either wild-type SINV AR86, one of the above-described nsP1 mutants, or mock infected. At 8 h postinfection, the cells were lysed via the addition of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5]–50 mM NaCl–1% [vol/vol] Nonidet P40 [NP-40]–0.5% [wt/vol] SDS–0.05% [wt/vol] sodium deoxycholate–1 mM EDTA) followed by vigorous vortexing prior to storage at −80°C until further use. Equal amounts of whole-cell lysates were resolved using SDS-PAGE and transferred to nitrocellulose membranes for downstream immunodetection. The resulting blots were probed for anti-SINV nsP2 polyclonal sera (a gift from R.W. Hardy at Indiana University−Bloomington) and anti-actin (clone mAGGEa; ThermoFisher) and probed with the appropriate horseradish peroxidase (HRP)-labeled secondary antibodies using the iBind Flex Western system with HRP detection/blotting reagents (ThermoFisher). Detection of the SINV nsP2 and host actin proteins was accomplished via chemiluminescence with SuperSignal West Pico Plus chemiluminescent substrate (34579; ThermoFisher) and detected by an Azure C200 Imaging Station (C200; Azure Biosystems).
3
Western Blot Analysis of CAP1 and GAPDH
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The western blotting procedure was previously described [13 (link)]. Membranes were incubated with rabbit anti-CAP1 mAb (EPR8339(B); Abcam) or rabbit anti-GAPDH pAb (Santa Cruz Biotechnology), with a dilution of 1:1000 (CAP1) and 1:100 (GAPDH), the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit antibody) was added (at a dilution of 1:2000), and an incubation was performed for 3 h using the iBind Flex Western System (Thermo Fisher Scientific). Protein bands were detected with the enhanced Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) using LAS-3000 (Fujifilm).
4
Quantitative Protein Analysis via SDS-PAGE and Western Blotting
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The protein concentration in crude protein was determined with a BCA kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. We then boiled the samples for 10 min in loading dye (125 mM Tris/HCl, 10% SDS, 0.25% BPB, 10% 2-mercaptoethanol, 50% glycerol), and loaded 60 μg of the crude extract onto 12% acrylamide gels for separation by SDS-PAGE. Primary (anti his-tag mouse monoclonal antibody at a concentration of 1:500) and secondary (goat anti-mouse IgG, HRP-conjugated at a concentration of 1:2000) antibodies were used for the Western blotting test via the iBind™Flex Western System (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 4 h, following the manufacturer’s protocol. After washing with ddH2O solution three times, CDA target bands were visualized by incubating the membranes in ECL reagents with the ChemiDoc XRS Imaging System and the Image Lab 6.0 software (Bio-Rad Lab, Hercules, CA, USA).
5
Protein Extraction and Western Blotting
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hdLECs were washed three times with 1X PBS and 1X RIPA buffer, and 100 X phosphatase inhibitor (Pierce™, Thermoscientific) was added. A cell scraper was used to scrape the hdLECs off the 6-well plate. hdLECs were pipetted and submerged in the lysis RIPA solution. The lysate was collected in a 1.5-ml protein LoBind tube and incubated for 15 min in ice on an orbital shaker (Belly button, IBI Scientific) for complete lysis. The lysates were then centrifuged for 20 min at 18,300 RPM at 4℃, and the supernatant was collected in a separate Protein LoBind tube without disturbing the pellet. A bicinchoninic acid assay (BCA assay, Pierce™, Thermo Fisher) was performed to calculate protein concentration according to manufacturer’s instructions, and 25 μg of the total protein was used to perform the Western blot. The Invitrogen™ mini gel tank apparatus was used to run the Western blot on a 4%–12% Blot™ Bis Tris Plus polyacrylamide gel. Protein transfer to a PVDF membrane was carried out using an iBlot 2 dry blotting system (Lifesciences, Thermo Fisher) under the P0 module. The iBind Flex Western System (Thermo Fisher Scientific) was used to process the mini blots using an incubation time of > 2.5 h, after which the blots were imaged using a Bio-Rad ChemiDoc imaging system.
6
Quantifying STAT3 Activation and Localization
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Cytosol and nuclear content of STAT3 and its two phosphorylated active forms pSTAT3(Tyr705) and pSTAT3(Ser727), and mitochondrial content of pSTAT3(Ser727) and the Complex I subunit GRIM-19 (gene associated with retinoid-IFN-induced mortality) were determined by immunoblotting analyses. Extracts were heated at 70°C in equal volumes of x4 Protein Sample Loading Buffer. Twenty μg of protein were loaded per lane on a 10% Bis-Tris gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. Immunoblotting was performed using the IBind Flex Western System (Thermo Fischer Scientific, Waltham, MA) that applies sequential lateral flow to perform blocking and antibody binding. Loading control proteins were concomitantly probed: β-actin as loading control in cytosol and nuclear extracts; and voltagedependent anion channel-1 (VDAC-1) as loading control in mitochondrial extracts. For all immunoblotting, the Odyssey LI-COR scanner and software (LI-COR Biotechnology, Lincoln, NE) were used for detection and quantitative analysis.
7
Protein Expression Analysis via SDS-PAGE and Western Blotting
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Polyacrylamide gel electrophoresis was performed on pre-cast NuPAGE™ 4–12% Bis-Tris Midi Protein Gels (Invitrogen) polyacrylamide gels. Protein samples analysed under reducing conditions were incubated with 250 mM DTT before loading onto the gel. Gels were electrophoresed in NuPAGE™ MES SDS Running Buffer (Invitrogen). Total protein in a gel was visualised by Coomassie staining (Quick Coomassie, Generon). Proteins were blotted on nitrocellulose membranes using the BioRad TransBlot Turbo Transfer System (BioRad). Different mouse and rat antibodies at a concentration of 1 µg/ml were bound to the membrane using the iBind Flex western system (Invitrogen), alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, A8438, 1:1000) or alkaline phosphatase-conjugated goat anti-rat IgG (Sigma, A8438, 1:1000) were used for detection. Western blots were developed using SIGMAFAST™ BCIP®/NBT (Sigma).
8
Western Blot Analysis of Protein Expression in THP-1 Macrophages
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For total protein extraction, THP-1-derived macrophages were rinsed with cold PBS and then lysed with cold RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl (pH 7.5)], supplemented with protease and phosphatase inhibitor cocktail mixtures. Protein concentrations were determined using the DC protein assay kit. Total proteins (20 µg/lane) were resolved by 4-20% SDS-PAGE and transferred to Immobilon-FL polyvinylidene difluoride membranes (Merck KGaA). Western blot analysis was then performed at room temperature for ~3 h using the iBind Flex Western system (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Briefly, primary antibodies against Nrf2 (1:1,000), GAPDH (1:8,000), totaland p-ERK (1:2,000 and 1:1,000, respectively), total and p-p38 (1:2,000 and 1:1,000, respectively), total and p-JNK (both 1:1,000), and IRDye680RD- or IRDye800RD-conjugated secondary antibodies (1:4,000 and 1:3,000, respectively) were diluted in iBind Flex FD Solution. Fluorescent blot imaging was performed using Odyssey CLx Imaging system (LI-COR Biosciences). Densitometric analysis was performed using Image Studio Lite software v4.0 (LI-COR Biosciences).
9
Western Blot Analysis of CD80, CD86, and CTLA4
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Samples from supernatants of transfected or infected cells were treated either directly or after a 20-fold concentration using a Vivaspin 20 30,000-molecular-weight-cutoff (MWCO) concentrator (Sartorius). Twenty-five microliters of sample was prepared in Laemmli buffer with (reducing condition) or without (nonreducing condition) 5% beta-mercaptoethanol. After electrophoresis on Criterion TGX 4 to 15% stain-free gels (Bio-Rad), the proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Transblot Turbo System). An IBind Flex Western system (Invitrogen) was used for the protein/antibody incubations and washes. Blots were probed either with 2.5 μg/ml of CD80-Fc, CD86-Fc, or CTLA4-Fc or with anti-Flag-HRP at a 1/1,000 dilution. For CD80-Fc, CD86-Fc, and CTLA4-Fc, an anti-human Fc-HRP antibody (Bethyl) at 1/3,000 was used as the conjugated antibody. The 1× iBind Flex Solution was used to block, dilute the antibodies, and wash and wet the iBind Flex Card. Immune complexes were detected using Amersham ECL Prime Western blotting reagents. Chemiluminescence was recorded with a ChemiDOC XRS molecular imager (Bio-Rad).
10
Dystrophin Protein Quantification in Muscle and Brain
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Protein was extracted from the tibialis anterior muscle or from the brain of 8-week-old male DmdEGFP mice and wild-type littermates using a combination of following two buffers (1:1) containing proteinase inhibitor cocktail: (i) 250 mM sucrose, 10 mM Tris-HCl, pH 8.2 and (ii) 20 % SDS, 20 % glycerol, 10 % β-mercaptoethanol, 12.5 % Western blot running buffer in bidest water; 50, 5, 0.5, or 0.05 μg protein were loaded on 3–8 % Tris-acetate gradient gels (Novex, Life Technologies), electrophoresed, and wet-blotted onto a nitrocellulose membrane. NuPage® LDS sample buffer (Invitrogen) was used for gel electrophoresis. The blots were probed using the iBind Flex® Western System (Invitrogen, LifeTechnologies) with mouse antibodies directed against the dystrophin rod domain (Dys1 or MANDYS19) and a rabbit polyclonal antibody against the dystrophin C-terminus (clone H4, self-made, gift from Cyrille Vaillend) together with secondary fluorescently labeled antibodies (AlexaFluor® 700 and 800). A second blot was probed with a monoclonal anti-GFP antibody together with a secondary fluorescent antibody (AlexaFluor® 700). As a loading control, a mouse monoclonal anti-vinculin antibody was used. Bands were visualized using the Odyssey CLx system (Li-Cor) (Fig. 1c ). All used antibodies are listed in Additional file 2 : Tables S1 and S2.
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