All Western data were generated from whole cell lysates. Cells were scraped into medium and pelleted by centrifugation along with the floating population, washed once with cold PBS, lysed with 2x Laemmli sample buffer (Bio-Rad Laboratories, Hercules CA, USA) and heated at 100 °C for 5 min. Proteins were quantified using the Pierce 660 Protein Assay kit (Thermo Fisher Scientific, #22662) and loaded onto 4-20% SDS polyacrylamide gels (Invitrogen). After electrophoresis, proteins were transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk in Tris Buffered Saline (TBS). Immunodetection was performed with the indicated primary antibodies for overnight incubation at 4 °C. Following washing and incubation with the appropriate HRP-conjugated secondary antibodies, signals were visualized by Supersignal West Dura Extended Duration substrate (#34076, Pierce, Rockford, IL, USA) or ProSignal Femto ECL reagent (#20-302, Genesee Scientific, El Cajon, CA, USA). All western analysis data are representative of at least three independent biological replications. Quantifications were done by ImageJ (https://imagej.nih.gov/ij/ ). After background subtraction, pSTAT3 data were normalized to STAT3 total protein and graphed as fold change with respect to untreated control from at least three independent experiments.
Pierce 660 protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
The Pierce 660 Protein Assay Kit is a colorimetric assay used for the quantification of protein concentrations in aqueous samples. The kit employs a reagent that forms a colored complex with proteins, which can then be measured using a spectrophotometer. The assay is compatible with a wide range of sample types and can be performed in a microplate format, making it suitable for high-throughput applications.
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5 protocols using pierce 660 protein assay kit
1
Western Blot Protocol for Quantifying pSTAT3
2
BMP-2-Specific Affibody Conjugation to HA
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BMP-2-specific affibodies were expressed in E. coli and purified as previously described.(41 (link)) HA-Nor-Ox (14.5 mg) was dissolved in 1.5 mL of 0.71 mg/mL affibody in PBS (1.45 × 10−4 mmol) to create a solution with 1:1 ratio of affibody to HA-Nor-Ox. 15 μL of 10% w/v Irgacure 2595 in methanol was added to the reaction vial, stirred, and illuminated with 365 nm light for 10 minutes. The solution was dialyzed against HEPES buffer pH 7.0 for 1 day and diH2O for 2 days. The affibody-conjugated solution of HA (HA-Nor-Ox-Affibody) was sterile filtered through a 0.22 μm filter, frozen at −80 °C, and lyophilized. The amount of affibody conjugated to the polymer was quantified by dissolving 2–3 mg of HA-Nor-Ox-Affibody in PBS at 2% w/v and determining the protein concentration of the solution using a Pierce 660 Protein Assay Kit (Thermo Fisher Scientific).
3
Synovial Fluid Collection and Processing
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Synovial fluids from RA and osteoarthritis (OA) patients were collected in collaboration with LIMIDRA (Luxembourg Immune-Mediated Inflammatory Disease Research Association) and CHU (Centre Hospitalier Universitaire) of Brest, France.
Synovial fluids from RA and OA patients were collected under written informed consent from all participants, following the good clinical and ethical practices approved by the Ethics Review Panel (ERP 17-005 NEUTRA, 14 June 2017) of the University of Luxembourg according to the guidelines of the “Comité National d’Ethique de Recherche” (CNER 201701/07, 28 February 2017) and the “Commission nationale pour la protection des données” (CNPD) from Luxembourg.
Synovial fluids were treated with 20 U/mL hyaluronidase (Carl Roth, Karlsruhe, Germany) for 30 min at 37 °C. Then, synovial fluids were filtered through 100 µm cell-strainers (Greiner Bio-One, Frickenhausen, Germany) centrifuged at 300 g for 15 min and treated with SigmaFast protease/phosphatase inhibitor cocktail (Merck, Darmstadt, Germany). The synovial fluids acellularity was checked using microscopy prior to determining the protein concentration using a Pierce 660 protein assay kit (Thermo Fisher Scientific, Erembodegem, Belgium). Finally samples were alicoted and stored at −80 °C.
Synovial fluids from RA and OA patients were collected under written informed consent from all participants, following the good clinical and ethical practices approved by the Ethics Review Panel (ERP 17-005 NEUTRA, 14 June 2017) of the University of Luxembourg according to the guidelines of the “Comité National d’Ethique de Recherche” (CNER 201701/07, 28 February 2017) and the “Commission nationale pour la protection des données” (CNPD) from Luxembourg.
Synovial fluids were treated with 20 U/mL hyaluronidase (Carl Roth, Karlsruhe, Germany) for 30 min at 37 °C. Then, synovial fluids were filtered through 100 µm cell-strainers (Greiner Bio-One, Frickenhausen, Germany) centrifuged at 300 g for 15 min and treated with SigmaFast protease/phosphatase inhibitor cocktail (Merck, Darmstadt, Germany). The synovial fluids acellularity was checked using microscopy prior to determining the protein concentration using a Pierce 660 protein assay kit (Thermo Fisher Scientific, Erembodegem, Belgium). Finally samples were alicoted and stored at −80 °C.
4
CenpA Quantification in RPE-1 Cells
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RPE-1 cells were lysed using cynase-compatible lysis buffer with a final composition of 1% Triton X-100, 25 mM NaCl, 50 mM Tris pH 8.0, 10 mM MnCl2, and 50 U/ml cynase endonuclease (RiboSolutions, Inc., Cedar Creek, TX). Cell lysates were analyzed for protein concentration using a Nanodrop ND-1000 Spectrophotometer and the Pierce 660 Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Aliquots were diluted in BRB80 and PAGE buffer. Samples were run on 15% SDS-PAGE gels and then transferred to PVDF membranes. Membranes were probed with primary antibodies for anti-CenpA (Cell Signaling Technology, Danvers, MA; catalog # 2186S; dilution 1:10,000), and anti-H4 (a gift from Dr. Judith Berman; anti-Histone H4 antibody dilution 1:5000), followed by a horseradish peroxidase-linked anti-rabbit secondary (Santa Cruz Biotechnology, Dallas, TX; catalog # SC2004) and then developed using chemiluminescence. For quantification, total CenpA intensity was normalized to total H4 intensity for each sample. See source data file for uncropped scans of the western blots.
5
BMP-2-specific Affibody Conjugation to HA
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BMP-2-specific affibodies were expressed in E. coli and purified as previously described.41 (link) HA-Nor-Ox (14.5 mg) was dissolved in 1.5 mL of 0.71 mg mL−1 affibody in PBS (1.45 × 10−4 mmol) to create a solution with 1 : 1 ratio of affibody to HA-Nor-Ox. 15 μL of 10% w/v Irgacure 2595 in methanol was added to the reaction vial, stirred, and illuminated with 365 nm light for 10 minutes. The solution was dialyzed against HEPES buffer pH 7.0 for 1 day and diH2O for 2 days. The affibody-conjugated solution of HA (HA-Nor-Ox-Affibody) was sterile filtered through a 0.22 μm filter, frozen at −80 °C, and lyophilized. The amount of affibody conjugated to the polymer was quantified by dissolving 2–3 mg of HA-Nor-Ox-Affibody in PBS at 2% w/v and determining the protein concentration of the solution using a Pierce 660 Protein Assay Kit (Thermo Fisher Scientific).
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