Toxoplasma gondii tachyzoites (RH strain) were maintained in BALB/c mice by intraperitoneal passages. Peritoneal exudates from 40 mice were harvested and washed twice (720 × g, 10 min, 4°C) in PBS supplemented with a protease cocktail inhibitor (10 mg/ml aprotinin, 50 g/ml leupeptin, and 1.6 mmol/L phenylmethylsulfonyl fluoride). To prepare soluble T. gondii antigen (STAg), the parasite suspension was lysed by five sonication cycles (60 Hz for 1 min each) on ice. After centrifugation (10,000 g, 2 h, 4°C) supernatants were collected and sterilized by filtration through a 0.2 μm-pore size membrane (Corning Costar Corp., Cambridge, MA). The protein concentration was determined by Bradford (Quick Start™ Protein Assay, Bio-Rad laboratories, Hercules, CA) and aliquots were stored at −80°C until use.
Quick start protein assay
Manufactured by Bio-Rad
Sourced in United States
The Quick Start protein assay is a colorimetric-based assay used to determine the concentration of protein in a sample. It provides a simple and accurate method for quantifying protein levels in a variety of biological samples.
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Lab products found in correlation
0.2 μm pore size membrane, by Corning (1 mentions)
Nucleotide removal kit, by Qiagen (1 mentions)
Neg502a, by PerkinElmer (1 mentions)
T4 polynucleotide kinase, by Takara Bio (1 mentions)
Image gauge software, by Fujifilm (1 mentions)
Imaging plate, by Fujifilm (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Las 4000 image analyzer, by GE Healthcare (1 mentions)
Vivaspin 20, by Sartorius (1 mentions)
Emulsiflex b15, by Avestin (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Sh 9000, by Corona Electric (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
10 protocols using quick start protein assay
1
Preparation of Toxoplasma gondii Antigen
2
Tyrosine-Linked DNA Substrate Assay
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All single-standard DNA substrates share the commons sequence of 5′-TCCGTTGAAGCCTGCTTT-3′. Oligonucleotides were synthesized in the reversed direction, as described previously (42 (link)). CBV was prepared according to the literature (43 (link)). Total cell lysates from DT40 Tdp1−/− and Tdp1−/−hTDP1 cells were prepared in the same manner as previously described (44 (link)). The protein concentration of lysates was determined by Bradford protein assay (Quick Start protein assay, Bio-Rad Laboratories). To prepare DNA substrate, oligonucleotides with 3-phosphotyrosine and CBV linkages synthesized were incubated with [γ-32P]adenosine triphosphate (NEG502A, PerkinElmer Life Sciences) and T4 polynucleotide kinase (Takara) for 5′-end labeling. After being purified using a nucleotide removal kit (Qiagen), 1 nM labeled oligonucleotide was incubated with cell lysates at 25°C for 15 min. Samples were separated by 16% denaturing polyacrylamide gel electrophoresis (7 M, urea). Dried gel was exposed on the imaging plate (Fujifilm) and then scanned in Fuji Bas 2500 system. Quantification was performed by Image Gauge software (Fujifilm).
3
Protein Concentration Measurement by Bradford Method
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Example 4
The concentration of a protein was measured by a Bradford method. In the Bradford method, Quick Start Protein Assay (Bio-Rad Laboratories, Inc.) was used, and the protein concentration was calculated based on a standard curve drawn using bovine γ-globulin as a standard protein.
The results of measurement of protein concentration are shown in
4
Protein Extraction and Immunoblotting
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Cells were lysed in NET buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 1 mM EDTA) supplemented with 1 mM DTT, 1 mM PMSF, 10 μg/ml DNase, 10 μg/ml RNase, 10 mM sodium butyrate (class-I, II KDAC inhibitor), and 20 mM nicotinamide (class-III or sirtuin KDAC inhibitor) by exposure to high pressure using an EmulsiFlex-B15 (Avestin). After removing the cell debris by centrifugation, the cleared lysates were concentrated with a Vivaspin 20 (Sartorius). The protein concentration was measured with the Quick start protein assay (Bio-Rad). Lysate aliquots containing 20 μg of protein were separated by 10% SDS-PAGE and then transferred to an Immobilon-P membrane (Millipore) using a semidry apparatus. The blot was blocked with 3% (w/v) skim milk in TBST, and then incubated with a mixture of rabbit polyclonal antibodies (1:1000 each in 3% [w/v] milk-TBST) as the primary antibody at 4°C overnight. The blot was then incubated with an horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 in 3% milk-TBST; Sigma) for 1 h at room temperature. Antigens on the membranes were detected with an LAS4000 image analyzer (GE Healthcare).
5
Nrf2 Protein Extraction and Analysis
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Protein was extracted using 1 × RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor (Roche Diagnostics K.K, Tokyo, Japan), phosphatase inhibitor cocktail (Sigma Aldrich), and 1 mM PMSF (Thermo Scientific, Waltham, MA, USA). With nuclear factor (erythroid-2-related factor)-2 (Nrf2) protein extraction, 10 μM MG132 (Sigma Aldrich) was additionally added to the above extraction buffer. Protein concentration was determined using a Quick Start protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and 25 μg of protein was used in each SDS-PAGE run. 7.5% Mini-Pro TEAN Precast Gel (Bio-Rad) was used for each analysis. Protein extracts were transferred to a PVDF membrane. After blocking for 1 h, the membrane was incubated with primary antibodies (anti-Nrf2, 1:200, #14596, Cell Signaling) overnight at 4 °C. After washing, the membrane was incubated with secondary antibodies (anti-rat IgG, sc-2032, 1:5000, Santa Cruz) for 1 h at room temperature. Expression of β-actin (1:5000, sc-47778, Santa Cruz) was used as an internal control.
6
Quantification of Hepatic p53 Levels
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Each mouse liver was homogenized in nine volumes of low-salt RIPA buffer and the homogenate was centrifuged at 10,000 ×g for 10 min at 4°C. Protein levels of p53 in the supernatant were quantified using a p53 Enzyme Immunometric Assay kit (Assay Designs, Ann Arbor, MI, USA). Total protein concentration was measured using the Quick Start Protein Assay (Bio-Rad, Hercules, CA, USA), to normalize p53 levels.
7
Quantifying Skin Barrier Cytokine Levels
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The 24 mm × 60 mm strips of tape with adhered stratum corneum samples were cut into ~5‐mm2 pieces and immersed in 1 mL phosphate‐buffered saline (PBS) and sonicated for 30 seconds on ice to obtain the extract. Samples were then centrifuged at 4°C and 16 100 g for 10 minutes to eliminate the insoluble fraction, and the supernatant was used for further analysis as the stratum corneum extract. Total protein content in supernatant was measured using a Quick Start protein assay (Bio‐Rad Laboratories), with bovine serum albumin (BSA) used as a reference. The levels of IL‐1α, IL‐1ra, and IL‐10 in the supernatant were measured using respective ELISA kits (R&D Systems). The amount of target proteins per total protein content was calculated, and the IL‐1ra/IL‐1α ratio was calculated, as previously described.19
8
Extracellular ATP Quantification in Na2S-Treated Cells
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The cells were plated at a density of 1.5 × 10 4 cells/cm 2 on 24-well plates. Once the cells reached confluence, they were incubated with ACSF (37°C) for 1 h before Na2S (final concentrations, 0.001-150 μM) was added. Na2S was applied by dropping a highly concentrated solution. Five minutes later, samples (50 μl) were collected and boiled.
Remaining cells were suspended in 0.1 N NaOH and sonicated. The protein content of cell lysates was measured using the Quick Start protein assay (Bio-Rad, Hercules, CA). ATP in the samples was measured with the luciferin-luciferase technique using the ATP Determination kit (Invitrogen) and a microplate reader (SH-9000; Corona Electric). A calibration curve was prepared using standard solutions containing known concentrations of ATP, and concentrations of ATP in the samples were calculated from it. The amounts of ATP were expressed as the extracellular amount per milligram of protein in cell lysates (pmol/mg protein).
Remaining cells were suspended in 0.1 N NaOH and sonicated. The protein content of cell lysates was measured using the Quick Start protein assay (Bio-Rad, Hercules, CA). ATP in the samples was measured with the luciferin-luciferase technique using the ATP Determination kit (Invitrogen) and a microplate reader (SH-9000; Corona Electric). A calibration curve was prepared using standard solutions containing known concentrations of ATP, and concentrations of ATP in the samples were calculated from it. The amounts of ATP were expressed as the extracellular amount per milligram of protein in cell lysates (pmol/mg protein).
9
Quantifying MAPK Phosphorylation in Plant Cells
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Detection of phosphor-MAPK was performed as described previously (Singh et al. 2012) , with the following modifications. Phosphorylation of MAPK were determined in crude protein extracts from cultured rice cells or Arabidopsis cells treated with 1 µM elicitors for 5 or 10 min and were prepared as described previously (Romeis et al. 1999) . The protein concentration was determined using the Quick Start protein assay (Bio-Rad) with BSA as a standard. Crude extracts (10 µg total protein per lane) were separated on a 12.5% sodium dodecyl sulfate gel, and proteins were transferred onto nitrocellulose membrane (GE Healthcare) by semidry electroblotting (Bio-Rad). Phosphorylated MAPK were detected by overnight incubation with anti phospho-p42/44 MPK primary antibodies (1:2,000) (Cell Signaling Technology), followed by incubation with antirabbit immunoglobulin G (H+L chain)-horseradish peroxidase secondary antibodies (MBL) for 30 min. The signals were visualized using ECL Plus Western blotting detection system (GE Healthcare), following the manufacturer's instructions.
10
Cytokine and Chemokine Quantification in Rat
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The plasma albumin concentration was measured using the Rat Albumin ELISA kit (AKRAL-120, Shibayagi, Gunma, Japan). Cytokines (except for TGF-β 1, 2 ) and chemokines were measured in colon tissue homogenates and spleen cell cultures using the Milliplex Rat Cytokine/Chemokine Panel (RCYTO-80K; Millipore, Billerica, MA). TGF-β 1, 2 in colon tissue homogenates and spleen cell cultures were measured using ELISA kits (TGF-β 1 , MB100; TGF-β 2 , DB250; R&D Systems, Abingdon, UK). CD11b (macrophage marker, also known as integrin αM) in colon tissue homogenates was measured using ELISA kit (SEB685Ra; Cloud-Clone, Houston, TX). Protein quantification in colon homogenate was done using Quick Start Protein Assay (Bio Rad, Hercules, CA).
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