Whole-cell lysates were made from monolayers of cultured cells in the exponential phase of growth. Cells were subjected to various treatment protocols (as indicated in the figure legends) after seeding into tissue culture flasks at 30–40% confluence. Cells were harvested at appropriate time points using a non-enzymatic cell dissociation fluid, washed twice in PBS and resuspended in RIPA buffer. Whole-cell lysate protein was loaded onto SDS-PAGE gels, electrophoresed, and Western transferred to a PVDF membrane. Probing of the membranes for GAPDH levels to act as loading controls was carried out by membrane stripping with 1 mM sodium azide in PBS for 1 h, followed by incubation using a murine antibody (Sigma Aldrich, St. Louis, MI, USA). Visualisation was carried out using alkaline phosphatase-conjugated secondary antibody (WesternBreeze®, Life technologies, Paisley, UK) with chemiluminescent substrate (CDP Star®, Invitrogen, Carlsbad, CA, USA). Protein expression levels were quantified by use of densitometry using chemiluminescent film, and processing of the scanned images as JPEG files was performed using Quantiscan 3.0 software (Biosoft®). Band intensity was normalised to GAPDH and was expressed relative to MCF-7/T47D controls (values shown are the means ± SD of three independent experiments, with p-values shown in the legends).
Cdp star
Manufactured by Thermo Fisher Scientific
Sourced in United States
CDP-Star is a chemiluminescent substrate used for the detection of alkaline phosphatase in western blotting and other immunoassay applications. It is designed to provide a sensitive and quantitative signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase linked anti flag m2 monoclonal antibody, by Merck Group (2 mentions)
Mouse anti strepii polyclonal antibody, by Qiagen (2 mentions)
Murine antibody, by Merck Group (1 mentions)
Western breeze, by Thermo Fisher Scientific (1 mentions)
Quantiscan 3, by Biosoft (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Sds sample buffer, by New England Biolabs (1 mentions)
Human thrombopoietin quantikine elisa kit, by R&D Systems (1 mentions)
Sample buffer, by Bio-Rad (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Mab5404, by Merck Group (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
T per, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Hyperfilm, by Cytiva (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Hybond p polyvinylidene fluoride pvdf membranes, by GE Healthcare (1 mentions)
Hyperfilm, by GE Healthcare (1 mentions)
24 protocols using cdp star
1
Whole-Cell Lysate Preparation and Western Blot Analysis
2
Western Blot Analysis of Hfq Protein
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Protein samples were generated by collecting the pellet for 1 ml of cells at OD600 ~ 1.0 and lysing with 100 µl 1X SDS sample buffer (New England Biolabs) and DTT by boiling for 10 min. The protocol is based on that described previously (28) . Samples (10 µl) were resolved on a 12% bis-Tris polyacrylamide NuPAGE gel (Invitrogen, USA) in 1X of 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (Invitrogen, USA) for 35 min at 180V. The gel was transferred using the iBlot2 (ThermoFisher Scientific) to a nitrocellulose membrane, blocked with 3% milk in 1X PBST (PBS with 0.1% Tween 20) and treated with primary anti-Hfq antibody, diluted 1:5000 in 0.3% milk in 1x PBST; the anti-Hfq antibody was preadsorbed with cell extract from a strain deleted for hfq. After washing, the membrane was incubated with 1:1000 dilution of goat anti-rabbit AP-linked secondary antibody (Cell Signaling) in 0.3% milk in 1XPBST at room temperature for 1hr, washed and developed using CDPStar (Invitrogen).
3
Automated TPO Quantification via CLEIA
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TPO-CLEIA is a one-step sandwich-type assay. The analyte, TPO, is captured by paramagnetic microparticles coated with rTN1. The analyte–microparticle complex was detected using ALP-labeled a020-D4. Assay calibrators ranged from 0 to 700 pg/mL (0, 20, 70, 200, and 700 pg/mL).
In the first step, 110 μL of anti-TPO antibody-coated microparticles, 40 μL of the sample, and 100 μL of ALP-labeled anti-TPO antibody were mixed and incubated for approximately 10 min. Then, the bound and free fractions were separated, an immunocomplex made of anti-TPO antibody-coated microparticles and TPO and ALP-labeled anti-TPO antibody was incubated with 100 μL of chemiluminescent substrate solution (CDP-Star; Applied BioSystems, Bedford, MA, USA) for 2.7 min, and the luminescence was measured using a luminometer in the STACIA CLEIA system (LSI Medience Corporation, Tokyo, Japan). The assay was fully automated, and measurements were completed within 20 min with a throughput maximum of 270 tests/h. A schematic representation of TPO measurement is shown inFigure 1 .
Plasma TPO concentrations measured by TPO-CLEIA were compared to those obtained by TPO-ELISA (Human Thrombopoietin Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA), a commercially available kit [23 (link)].
In the first step, 110 μL of anti-TPO antibody-coated microparticles, 40 μL of the sample, and 100 μL of ALP-labeled anti-TPO antibody were mixed and incubated for approximately 10 min. Then, the bound and free fractions were separated, an immunocomplex made of anti-TPO antibody-coated microparticles and TPO and ALP-labeled anti-TPO antibody was incubated with 100 μL of chemiluminescent substrate solution (CDP-Star; Applied BioSystems, Bedford, MA, USA) for 2.7 min, and the luminescence was measured using a luminometer in the STACIA CLEIA system (LSI Medience Corporation, Tokyo, Japan). The assay was fully automated, and measurements were completed within 20 min with a throughput maximum of 270 tests/h. A schematic representation of TPO measurement is shown in
Plasma TPO concentrations measured by TPO-CLEIA were compared to those obtained by TPO-ELISA (Human Thrombopoietin Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA), a commercially available kit [23 (link)].
4
Western Blot Protein Detection
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Total cell extracts were subjected to SDS-PAGE (77 (link)). Proteins were transferred onto a nitrocellulose membrane and probed using anti-FLAG (1:10,000), anti-FlgE2, or anti FliA (1:30,000) immunoglobulins (78 (link), 79 ). Detection was done with a secondary antibody conjugated to alkaline phosphatase and developed with CDPStar (Applied Biosystems).
5
Pseudovirus Production and Titration
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Pseudovirus (PSV) was prepared by co-transfection of HEK 293T cells with 2.5 µg of env and 5 µg of pSG3ΔEnv plasmids with PEI transfection reagent at a 1:3 DNA: PEI ratio. Culture medium was harvested 48 h later, filtered through a 0.22 µm pore sized filter and FBS was adjusted to 20% and stored at − 70 °C. The concentration of PSV was determined by a chemiluminescent ELISA (Aalto Bio-reagents) and TROPIX® detection system (CDP-Star®, Applied Biosystems). PSV titre was determined by infecting TZM-bl cells for 48 h with known p24 concentration and relative luminescence units (RLU) were compared between Env pseudotyped virus and pSG3ΔEnv PSV infected cells (background) and a titre resulting in 50 X RLU above background was used for MDDC stimulation.
6
Analyzing Protein Nitrotyrosination in Tissues
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Animal tissues were harvested in T-PER (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor (Sigma-Aldrich), were homogenized with a polytron on ice, and were centrifuged at 12,000×g for 20 min at 4 °C. Supernatants were collected and protein concentrations were measured with the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, CA, USA). Protein samples (45 μg) were mixed with 2× sample buffer (Bio-Rad Laboratories), were heated at 100 °C for 5 min and then were electrophoresed onto 4–20% pre-cast gels (Bio-Rad Laboratories) followed by transblotting to nitrocellulose membranes (0.45 μm). Membranes were rinsed in TTBS (Tris-HCl with NaCl and Tween 20) and were incubated in 5% blocking buffer (blotto in TTBS, Santa Cruz) for 1 h at room temperature before being probed with primary antibody (mouse anti-nitrotyrosine, MAB5404, 1:1000 in 1% blotto; Chemicon, now Millipore) overnight at 4 °C. After washing 3× with TTBS, membranes were incubated in alkaline phosphatase (AP) conjugated-secondary antibody (1:5000 in 1% blotto, Promega) at room temperature for 1 h. Membrane blots were washed 3× with TTBS followed by 2× assay buffer (1×) and then were incubated in substrate solution (CDP-Star, Applied Biosystems, now Thermal Fisher) for 10 min. The signal intensity of the protein bands was measured by employing Image Studio Lite software (LI-COR, Lincoln, NE, USA).
7
Measuring Plasma sCLEC-2 Levels by CLEIA
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We measured the plasma sCLEC-2 levels by a chemiluminescent enzyme immunoassay (CLEIA) using previously described monoclonal antibodies and the STACIA CLEIA system (LSI Medience, Tokyo, Japan). In brief, magnetic particles were coated with the anti-sCLEC-2 monoclonal antibody 5D11. The plasma samples were then incubated with antibody-coated magnetic particles, and after washing, they were incubated further with the alkaline-phosphatase-labeled anti-sCLEC-2 monoclonal antibody 11E6. After washing again, the magnetic particles were incubated with chemiluminescent substrate solution (CDP-Star; Applied BioSystems) and the luminescence was measured using the luminometer installed in the STACIA system. The D-dimer levels were determined via the latex agglutination method using LPIA-GENESIS D-Dimer (LSI Medience).
8
SDS-PAGE Protein Detection Protocol
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Cell pellets were boiled in SDS-reducing buffer for 20 min and centrifuged at 14,000 x g for 10 min at RT to obtain the supernatant. Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes. Affinity tagged proteins were detected on membranes by immunoblotting with alkaline phosphatase-linked anti-Flag M2 monoclonal antibody (Sigma) and with mouse anti-StrepII polyclonal antibody (QIAGEN) followed by goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma). Alkaline phosphatase activity was detected by chemiluminescence using CDP-Star (Applied Biosystems) with X-ray film (Hyperfilm; Amersham Biosciences).
9
Immunoblotting for Protein Detection
10
Haloarchaeal Protein Expression Analysis
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Hfx. volcanii strains were grown in complex medium to stationary-phase (OD600 of 3.0-3.5 in 4 ml ATCC 974 in 13 × 100 mm tubes), harvested by centrifugation (14,500 × g, 2 min at room temperature). Cell pellets were boiled in reducing SDS buffer (2% w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol, 0.002% w/v bromphenol blue and 62.5 mM Tris–HCl, pH 6.8). Proteins were separated by 12% SDS-PAGE and transferred to Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare Bio-Sciences, Piscataway, NJ) at 4°C for 2.5 h at 90 V by tank blot. Ponceau Red S Stain was used to confirm equal transfer (Boston Bioproducts, Ashland, MA). StrepII-tagged proteins were detected using unconjungated rabbit anti-StrepII polyclonal antibody (Genscript USA, Piscataway, NJ) and alkaline phosphatase (AP)-linked goat anti-rabbit IgG (H + L) antibody (SouthernBiotech, Birmingham, AL), as primary and secondary antibodies respectively. AP signals were detected by chemiluminescence with CDP-Star according to supplier’s protocol (Applied Biosystems, Carlsbad, CA) and visualized with X-ray film (Hyperfilm, GE Healthcare Bio-Sciences). Equivalent protein loading was further confirmed by staining samples similarly separated by SDS-PAGE in gel with Coomassie Blue R-250.
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