Total cellular protein (15 µg) was separated on an 8.5% Tris-HCl Criterion 12-well gel (Mini-PROTEAN; Bio-Rad, California, USA) at 200V for 30 min. Protein transfer from gels to PVDF membranes was carried out using a semi-dry system (Bio-Rad) set at 250 V, 0.1 A for 30 min. Rabbit source primary antibodies (×1000) against TCF7L1 (#2883), PARP (#9532), c-PARP (#5625), β-catenin (#8480), Cyclin D1 (#55506), c-Myc (#5605) and GAPDH (#5174) were obtained from Cell Signal Technology (Tokyo, Japan). Blots were developed using Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan) and visualized with HRP-Goat Anti-Rabbit IgG (JIRL, Pennsylvania, USA) using the ImageQuant 800 system (Cytiva, Massachusetts, USA). Protein expression was quantified using NIH’s ImageJ software. There were three duplicates of each experiment.
Imagequant 800 system
Manufactured by Cytiva
Sourced in United States
The ImageQuant 800 system is a compact, high-performance imager designed for analyzing fluorescent and chemiluminescent signals on various gel and blot formats. It features a charge-coupled device (CCD) camera and multiple excitation sources to capture images of biomolecules, such as proteins and nucleic acids, labeled with fluorescent dyes or detected using chemiluminescent substrates.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Tcf7l1, by Cell Signaling Technology (1 mentions)
C parp, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Semi dry system, by Bio-Rad (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (1 mentions)
Total ampk, by Cell Signaling Technology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
Novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
14 protocols using imagequant 800 system
1
Protein Expression Analysis by Western Blotting
2
Analyzing AMPK and p70S6K Signaling in ARPE Cells
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ARPE cells were collected by scraping into ice-cold PBS and centrifugation at 450xg for 5 min. The cells were lysed in buffer (100mM NaPi, pH 7.5, 150mM NaCl, 2mM EDTA and 1% Igepal, with protease inhibitors) for 15 min at 4°C and centrifuged at 20,000 xg to remove insoluble debris. For detection of AMPK and p70S6 kinase, phosphatase inhibitors were added to the lysis buffer. Protein concentration was determined using a BCA assay kit (ThermoFisher). Approximately 20μg protein was resuspended in NuPage LDS loading dye (ThermoFisher) containing 50mM DTT, heated at 70°C for 10 min, and subjected to electrophoresis and transfer using a Novex 4–12% BisTris gel (Invitrogen) in MOPS buffer as we have described (Chen et al., 2005 (link)). Blots were probed using primary antibodies against: NPC1 (ab134113; Abcam), total AMPK (5831; Cell Signaling Technology), AMPK phospho-Thr172 (2535; Cell Signaling Technology), p70S6 kinase (2708; Cell Signaling Technology), or p70S6 kinase phospho-Thr389 (9234; Cell Signaling Technology) and GAPDH (MAB374; EMD Sigma) and secondary anti-rabbit and anti-mouse antibodies conjugated with horseradish peroxidase (ThermoFisher). Proteins were visualized by chemiluminescence using SuperSignal West Dura substrate (ThermoFisher) according to the manufacturer’s recommendation as we have described (Chen et al., 2010 (link)) on an ImageQuant 800 system (Cytiva).
3
Quantitative Protein Analysis in EC Cells
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Total protein was extracted from EC cells using RIPA lysis buffer and a protease inhibitor cocktail (Applygen Technologies, Inc.). Protein concentrations were determined using a BCA Kit (Thermo Fisher Scientific, Inc.). A total of 50 µg protein/lane was electrophoresed via 10% SDS-PAGE gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. Blocking was performed using 5% skimmed milk for 1 h at room temperature. The following primary antibodies were used: Anti-ISG15 (catalog no. ab285367; Abcam), anti-tubulin (catalog no. 5335; Cell Signaling Technology, Inc.), anti-β-actin (catalog no. 3700; Cell Signaling Technology, Inc.) and anti-phosphorylated (p)-retinoblastoma-associated protein (RB1; Ser807/811) (catalog no. 8516; Cell Signaling Technology, Inc.); the antibodies and TBST (20% Tween) were diluted at a ratio of 1:1,000 and incubated overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (catalog no. 9003-99-0) were purchased from Applygen Technologies, Inc., diluted in 5% skimmed milk and incubated at room temperature for 1 h. After incubation with chemiluminescence solution (catalog no. WBKLS0050; Merck KGaA), the signals of the protein bands were acquired using an ImageQuant 800 system (Cytiva).
4
Quantitative Western Blot Analysis of Liver Fibrosis Markers
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Cells and liver samples were lysed and homogenized in Protease Inhibitor buffer (S8820, MilliporeSigma). Protein concentration was determined using Quick Start Bradford 1X Dye Reagent (5000205, Bio-Rad). Electrophoretically separated proteins were transferred to PVDF membranes (IPFL00010, MilliporeSigma) and incubated with the following primary antibodies: mouse anti–α-SMA (1:1000, MilliporeSigma, A5228), rabbit anti–collagen IV (1:1000, Abcam, Ab227616), mouse anti-GATA4 (1:1000, R&D Systems, MAB2606), rabbit anti-SMAD7 (1:100, Invitrogen, 42-0400), mouse anti-phosphoSTAT1 (Tyr701) (1:500, Invitrogen, 33-3400), rabbit anti-STAT1 (1:1000, Cell Signaling, 9172), rabbit anti-GAPDH (1:15,000, Cell Signaling, 2128), and mouse anti–β-actin (1:10,000, MilliporeSigma, A5441). Secondary antibodies rabbit anti-IRDye 800CW (LI-COR, 925-32211), mouse anti-IRDye 680RD (925-68070), mouse anti-IgG-peroxidase (MilliporeSigma A9044), and rabbit anti-IgG peroxidase (MilliporeSigma A0545) were used. The Odyssey CLx Infrared Imaging System (LICOR) or enhanced chemiluminescence detection reagents (GE Healthcare Bio-Sciences Corp.) in the Image Quant 800 System (Amersham) was used for imaging the blots.
5
Validating CsTBH-targeted Flavonoid Genes
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EMSA was performed to validate the results from Y1H assay for CsTBH-targeted flavonoid biosynthesis genes (4CL, FLS, F3H, and BZ1). For each gene, a 29 bp sequence containing the binding cis-element in the promoter of the gene was labeled with biotin. MBP-TBH recombinant proteins were expressed in the Escherichia coli BL21 (DE3) strain and then purified using amylose magnetic beads (New England Biolabs, Cat # E8035S). DNA gel mobility shift assay was performed using the EMSA kit (Beyotime, China, Cat # GS009) following manufacturer's protocol. Briefly, the DNA probes and proteins were coincubated in the reaction buffer at room temperature. Specific competitor (nonbiotin) and nonspecific competitor (mutated) probes were added in the reaction mixture for competition reaction. After incubation, the reaction mixture was separated by 6% (v/v) native polyacrylamide gel, and then, labeled DNA was detected using the Amersham ImageQuant 800 system. The primers are listed in Supplemental Table S7 .
6
Western Blot Analysis of Inflammatory Signaling
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Isolated Mo cultured at 500,000 cells/well in the presence of different stimuli were lysed using RIPA buffer 1% of phosphatase and protease inhibitors (Roche Diagnostics) with 10 min of sonication. Protein lysates were resolved in a 10% acrylamide (30% Acrylamide/Bis Solution 29:1, Bio Rad) gel with SDS and transferred to a nitrocellulose membrane (Fisher Scientific). Afterwards, membranes were blocked with 4% BSA (Sigma-Aldrich) in Tris-Buffered saline and incubated at 4 °C overnight with primary antibodies directed either to phospho-p65 (Cell Signaling ref 3039), anti-p65 (ab32536, E379, Abcam;), anti-caspase-1 (AF6215; R&D Systems), anti-Gasdermin D (69469, E5O4N; Cell Signaling), anti-IL-1β (12242, 3A6; Cell Signaling) or anti-GAPDH (FF26A/F9; BioLegend) following manufacturer’s specifications. Each primary antibody was used at a 1:1000 dilution. Subsequently, the membranes were washed and incubated at RT for 1 h with the respective anti-rabbit (31460; Thermofisher; dilution 1:2000), anti-goat (81–1620; Thermofisher; dilution 1:2000) or anti-mouse (31430; Thermofisher; dilution 1:5000) secondary antibodies. Chemiluminescence of protein band intensity was quantified by ImageQuant 800 system (Amersham) using the IQ800 V1.2.0 Software.
7
EMSA with CcpA DNA/RNA Probes
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The 16 nt dsDNA probe (see Supplementary Data 6 ) was purchased from IDT as two single-stranded oligonucleotides, with the sense strand labelled with IRDye800. The RNA probe (see Supplementary Data 6 ) was purchased from IDT, then labelled with IRDye800 (LI-COR, 929-80020) using the 5’ end labelling Kit (Vector Laboratories, MB-9001). The concentration of labelled RNA was determined by the absorbance of the dye at 780 nm. The DNA and RNA probes were resuspended in buffer containing 150 mM KOAc, 20 mM HEPES, pH 7.5, and annealed by heating up at 94 °C for 2 min and cooling down to 4 °C in 20 min. For the EMSA, a mixture in total 10 µL contained 150 mM KOAc, 20 mM HEPES, pH 7.5, 1 mM Mg(OAc)2, 0.5 µg poly(dI-dC) (non-specific competitor, Thermo Scientific, 20148E), 0.1 µM probe, and increasing concentrations (0–40 µM) of purified recombinant CcpA WT or T18DT33D phosphomimetic mutant. After 1 h of incubation on ice, the mixtures were loaded on 1% TBE-agarose gel and run in 1× TBE at 100 V for 1 h at 4 °C. Images were acquired using the Imagequant 800 system (Amersham) using the fluorescent IRlong channel.
8
Western Blot Detection of HTF Proteins
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To detect the HTF proteins, nitrocellulose membranes were blocked for 1 h in PBST (PBS with 0.1% Tween-20 and 5% skimmed milk powder) and incubated with the monoclonal anti-FLAG® M2-Peroxidase (HRP) antibody (1:5000; Sigma-Aldrich A8592) or the anti-TAP antibody (1:5,000 dilution; ThermoFisher CAB1001) overnight at 4 °C. After the anti-TAP incubation, membranes were washed twice for 10 min with PBST and incubated overnight with Goat anti-Rabbit IgG antibodies (1:5000; Invitrogen™ A16096). The membrane was washed three times with PBST before chemiluminescence. Membranes were subsequently incubated with the Pierce™ ECL Western Blotting Substrate (Thermo Scientific; 32106) and proteins were visualised using films or the ImageQuant 800 system (Amersham).
9
Western Blot Quantification of FTO, CDK6
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Total proteins were isolated from tissues and cells using RIPA lysis buffer mixed with phenylmethylsulfuryl fluoride (PMSF) (Bioss, China) and quantified using the bicinchoninic acid (BCA) method (Beyotime, China). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking by 5% skim milk, the PVDF membranes were incubated with an anti-FTO antibody (1:1 000, Abcam, USA), anti-CDK6 antibody (1:1 000, Abcam, USA) and anti-GAPDH antibody (1:5 000, Bioss, China) at 4 °C overnight. The PVDF membranes were then incubated with a secondary antibody (1:10 000, Boster, China) for 1 h at room temperature. The proteins were measured using the AMERSHAM ImageQuant 800 system (USA).
10
Peptide Concentration Characterization
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Protein concentration of the purified peptides was measured with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Germany) and the molar concentration was calculated and adjusted according to the molecular weight of the peptides (recAη-β MW: 13120.43; recAη-α MW: 15057.45). Peptides were stored until use in a − 80 °C freezer. For quality control, 1 μg of recombinant proteins and synthetic peptides were separated on a Tris-Tricine gel (10–20%, Thermo Fisher Scientific, Germany), stained with GelCode Blue stain, and imaged with an ImageQuant 800 system (Amersham, Germany).
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