Each sample was processed using cell lysis buffer with phosphatase and protease inhibitors (MilliporeSigma) and analyzed according to the assay protocol. The protein concentration of the cell lysate was measured using a BCA assay (G-Biosciences) and the MILLIPLEX MAP Multi-Pathway Magnetic Bead 9-Plex–Cell Signaling Multiplex Assay (48-681MAG; MilliporeSigma). The following probes were included in the kit: extracellular signal-regulated kinase/mitogen-activated protein kinase 1/2 (ERK/MAPK 1/2; Thr185/Tyr187), protein kinase B (Akt; Ser473), signal transducer and activator of transcription 3 (STAT3; Ser727), c-Jun N-terminal kinase (JNK; Thr183/Tyr185), ribosomal protein S6 kinase beta (p70S6K; Thr412), nuclear factor-kappa B (NF-кB; Ser536), signal transducer and activator of transcription 5 (STAT5A/B; Tyr694/699), cAMP-response element binding protein (CREB; Ser133), and p38 mitogen-activated protein kinases (p38; Thr180/Tyr182). The assay was carried out following the manufacturer's protocol, and fluorescence was assessed using xMAP technology on the MAGPIX platform (Luminex, Austin, TX). Mean fluorescent intensities were obtained in triplicate for each sample and normalized to total protein content.
Bca assay
Manufactured by G Biosciences
Sourced in United States
The BCA assay is a colorimetric detection method used to quantify total protein concentrations. It relies on the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the chelation of the Cu+ ions by bicinchoninic acid resulting in a purple-colored product that absorbs light at 562 nm.
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Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Magpix platform, by DiaSorin (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Fcap array version 3, by BD (1 mentions)
Cba human soluble protein flex set system, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Nexion 350x, by PerkinElmer (1 mentions)
Ultra 4 centrifugal filter, by Merck Group (1 mentions)
Anti mouse horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Semi dry transfer unit, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by G Biosciences (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Cary 300 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
10 protocols using bca assay
1
Cell Signaling Pathway Analysis
2
Quantifying Apoptosis Markers by Western Blot
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Western blot assays were used to identify the important molecular markers of apoptosis as previously described [40 (link)]. Briefly, SW620 cells incubated with DW-8 (10 μM, 30 μM for 24 h), the negative control (0 μM) and PTX (0.5 μM), were lysed using lysis buffer, M-PER™ (ThermoFisher, Rockford, IL, USA), along with a protease inhibitor cocktail containing Aprotinin, Destatin, E-64, Leupeptin, and Pepstatin A (Sigma-Aldrich Life Science, St. Louis, MO, USA). The protein concentration of the cell lysates was quantified using the bicinchoninic acid (BCA) assay (G-Biosciences, St. Louis, MO, USA) [41 (link)]. The lysates were loaded onto a polyacrylamide gel for electrophoretic separation. The proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked using 5% milk in Tris-buffer saline Tween 20 (TBST) for 1 h and incubated with primary antibodies (1:1000) at 4 °C with gentle shaking overnight. The membrane was washed with TBST and incubated with horseradish peroxidase labeled (HRP) secondary antibodies (1:4000) for 1 h. Subsequently, the membranes were washed and developed using enhanced chemiluminescent (ECl) substrates, SuperSignal™ Pico/Femto (ThermoFisher, Rockford, IL, USA). ImageJ software was used for the quantification of blots. Protein was normalized with beta-actin.
3
Protein Expression Analysis Protocol
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Protein isolation was done using RIPA buffer (Thermo Scientific-89901) and 1X PIC (Protease inhibitor cocktail cOmplete Roche 11697498001). The concentration of the protein was done using Bradford assay (Puregene PG-035-500ml) or BCA assay (G-Biosciences-786570). For western blotting 30ug protein was loaded in each well. 3% BSA in TBST was used as a blocking buffer. Primary antibodies: MHC (Invitrogen 14650382), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), PKM1(CST D30G6), PKM2 (CST D78A4), PDH (CST 3205), phospho-4EBP1 (CST2855), Total 4EBP1 (CST 9452), phospho-eIF4E (ab76256), Total-eIF4E (ab1126) were used in 1:1000 dilution and incubated at 4 °C overnight. Secondary antibodies: Anti-mouse IgG, HRP-linked (CST 7076) and Antirabbit IgG, HRP-linked (CST 7074) were used in 1:5000 dilution and incubated at room temperature for 1 h. The blots were developed using a developing solution (advansta-K-12049-D50).
4
Quantification of Biomarkers in Vitreous Humor
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Levels of βA1-crystallin and PTP1B in VH were measured by ELISA (βA1-crystallin: MyBiosource, USA; Cat# MBS7252187 and PTP1B: MyBiosource, USA; Cat# MBS761801) as per manufacturer’s instruction. Briefly, VH samples were centrifuged at 400 g for 15 min. The supernatant was homogenized by repeat pipetting and pulse vortexing (2 s X 5 times). Required volume of VH was diluted with 1X phosphate buffered saline (pH 7.4) or sample diluent, and ELISA was performed as per the manufacturer’s instructions. Absolute concentrations were derived based on standard curve. The levels of VEGF, IL-6, IL-8, and MCP1 in VH were measured by bead-based ELISA (Cytometric Bead Array, BDTM CBA Human Soluble Protein Flex Set System, BD Biosciences, USA) using a flow cytometer (BD FACS Canto II, BD Biosciences, USA). The beads and fluorescent signal intensities were acquired and recorded using BD FACSDiva software (BD Biosciences, USA). Standards were used to determine the absolute concentration of the analytes, and the calculations were performed using FCAP array Version 3.0 (BD Biosciences, USA). Total protein concentration in the VH was estimated by BCA assay (G-Biosciences) as per manufacturer’s protocol. The absolute concentrations (pg/ml) of the measured factors were normalized to the total protein concentrations (μg/ml) of the respective samples.
5
Gadolinium-Labeled Exosome Preparation
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Gadolinium-labeled exosomes were prepared by lipid insertion following membrane extrusion method 42 (link), 58 (link). In brief, different amounts of DSPE-DOTA-Gd (500, 1000, and 2000 µg) were solubilized in DPBS at 65 ºC. The GdL suspension was cooled to room temperature and co-incubated with 1mg of exosome protein for 30 min. The protein concentration was determined by BCA assay (G-Biosciences). The exosome-GdL (Exo-GdL) mixture was bath sonicated for 2 min and probe sonicated for 3 min with an amplitude at 30% and 30 s pulses on/off cycles on ice. The mixture was extruded through 100 nm pore size polycarbonate membrane to unify the hydrodynamic size. The Exo-GdL product was purified with an Amicon Ultra-4 centrifugal filter (Millipore, MA) with a molecular weight cut-off of 10 kDa. Rhodamine dye-labeled exosomes were prepared in the same fashion by hydrating 20 µg of L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (Ammonium Salt) (Egg Liss Rhod PE) film with 1 mg exosome protein. The amount of Gd on the exosomes was determined using inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer, NexION®350X). For ICP-MS, the Exo-GdL samples were digested with 2.0 mL of concentrated HNO3 for 5 h. After digestion, 100 μL of the sample was diluted with 10 mL of 2% HNO3 and analyzed using ICP-MS.
6
Western Blot Analysis of STAT1 and Phospho-STAT1 in RA-FLS
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The cell lysate was prepared from RA control and E2-treated RA-FLS through lysis in Radio-Immunoprecipitation Assay (RIPA) buffer (SIGMA) for Western blotting. Protein concentration was measured using BCA assay (G-biosciences) [45 (link)]. The 20 µg protein was resolved on 10% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane at 20 V for 50 min using a semi-dry transfer unit (Bio-Rad, Hercules, CA, USA) followed by overnight blocking at 4 °C with 5% bovine serum albumin (BSA) in 1X Tris-buffered saline-tween 20 (TBST) [44 (link)]. The membrane was then incubated with primary antibodies STAT1 (9172T, Cell Signaling), Phospho-STAT1 (Tyr701) (9167L, Cell Signaling) (dilution 1:5000) overnight at 4 °C followed by washing and incubation with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (dilution 1:8000) (Jackson, West Grove, PA, USA) for 1 h at room temperature. The band intensity was measured by chemiluminescence (ECL) reagent (Thermo Scientific, Rockford, IL, USA). The band intensity was normalized by the housekeeping protein (β-actin; sc-47778) as a loading control.
7
H2S Production Quantification in Tissues
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Measurement of H2S production was performed in liver and kidney homogenates according to a previously described protocol (Hine and Mitchell 2017 ). Briefly, 100 mg of flash-frozen liver and kidney were lysed in passive lysis buffer. Protein concentration was determined by BCA assay (G Biosciences, MO, USA) and 100 μg of protein lysate was loaded into 96-well plate. A 150-μL reaction solution containing 10 mM L-cysteine and 1 mM pyridoxal-5′-phosphate was added to the protein lysate. Filter paper that had previously been cut to the size of the plate, soaked in 20 mM lead(II)acetate trihydrate for 20 min, then dried under vacuum, was then securely attached to the plate. The assembled plate was incubated at 37 °C for 1 h. H2S sulphide gas produced during this time collects in the head space between the top of the solution in the well and the lead(II)acetate paper, forming a brown-black substrate on the paper. The amount of H2S present in each sample was subsequently quantified by densitometry analysis (ImageJ) of the brown-black substrate.
8
Western Blot Analysis of Testicular Proteins
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Total testicular protein was extracted using RIPA lysis buffer (G-Biosciences, St. Louis, USA) with 1X protease inhibitor cocktail (AMRESCO, USA). Protein concentration was determined using BCA assay (G-Biosciences, St. Louis, USA). The primary antibodies used were anti-YY1 alpha (Abcam, Cat. No. ab12132), anti-β-actin (Cell Signalling Technologies, Cat. No.4967L). Secondary antibodies used were anti-rabbit-horseradish peroxidase (HRP) conjugated (Epitomics, Cat. No.3053-1). Luminol and hydrogen peroxide were used as the substrate of HRP for developing the blot.
9
Quantification of Total Protein
10
Quantitative Assay of Liver H2S
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Measurement of H2S levels was performed in liver homogenates according to the previously described method [46 (link)]. Briefly, 100 mg of flash-frozen liver tissue was lysed in passive lysis buffer. Protein concentration was determined by bicinchoninic acid (BCA) assay (G Biosciences, MO, USA) and 100 ug of protein was loaded onto a 96-well plate. 150 uL of reaction solution containing 10 mM L-cysteine and 1 mM pyridoxal-5′-phosphate was added to the protein. Filter paper that had previously been cut to the size of the plate, soaked in 20 mM lead (II)acetate trihydrate for 20 min, then dried under vacuum, was then securely attached to the plate. The assembled plate was incubated at 37°C for 1 hr. H2S sulfide gas produced during this time collects in the head space between the top of the solution in the well and the lead (II)acetate paper, forming a brown-black substrate on the paper. The amount of H2S generated in each sample was quantified by densitometry analysis of the brown-black substrate (ImageJ).
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