Protein concentrations in nuclear extracts were quantified using the Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific). Bovine serum albumin (BSA) standards ranging in concentration from 0 to 2000 µg/ml were prepared following the manufacturer’s instructions. A total of 5 µl of each standard or sample was pipetted in duplicate into the wells of a 96-well plate followed by 250 µl Coomassie reagent. Well contents were mixed on a plate shaker for 30 s, and the plate was incubated at room temperature for 5 min, protected from light. Absorbance was measured at 595 nm using a Fluostar Omega microplate reader, and the concentration of protein in each unknown sample was extrapolated from a second polynomial curve generated using Mars data analysis software.
Coomassie bradford protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom
The Coomassie (Bradford) Protein Assay Kit is a colorimetric assay used to quantify the total protein concentration in a sample. It relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Clean up kit, by GE Healthcare (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Hmgb2, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Flexstation 3, by Molecular Devices (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Xmark microplate absorbance spectrophotometer, by Bio-Rad (2 mentions)
Phosphate buffer saline (pbs), by Thermo Fisher Scientific (2 mentions)
Rabbit monoclonal anti lrp1 antibodies ab92544, by Abcam (2 mentions)
Stripping buffer, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (2 mentions)
Irdye 680rd goat anti mouse igg h l, by LI COR (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
71 protocols using coomassie bradford protein assay kit
1
Quantifying Protein Concentrations in Nuclear Extracts
2
Muscle Lipid Peroxidation Quantification
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Using a commercially available kit (Northwest Life Science Specialties, Vancouver, WA, USA), muscle malondialdehyde (MDA) concentration, a marker of lipid peroxidation, was measured as previously described [16 (link)]. Briefly, cryopulverized muscle powder was diluted 1 mg tissue (wet weight) to 10 μL of assay buffer then sonicated while on ice and centrifuged at 11,000× g for 10 min at 0 °C. The supernatants were collected and stored at −80 °C until analysis. Samples were analyzed in triplicate with intra- and inter-assay CV of 2.2% and 1.6%, respectively. Malondialdehyde concentration was normalized to total protein quantified by the Coomassie Bradford Protein Assay kit (Thermo Fisher Scientific).
3
Cytokine Response in Hippocampus and Cortex
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Mice were subjected to three blocks of three training trials, and the next day, they were decapitated to obtain the cytokine data associated with the WM 18-h data. We used six young mice and seven adult mice that did not participate in the WM task and six young mice and seven adult mice that were trained on the WM task 18 h prior (WM 18-h condition). After the animals were deeply anesthetized and decapitated, the bilateral hippocampi and cortexes were removed and homogenized in ice-cold lysis buffer containing 25 mM HEPES, pH 7.43, 0.1% [(3-cholamidopropyl)dimethyl-ammonio]1-propanesulfonate, 5 mM MgCl2, 1.3 mM EDTA, 1 mM EGTA, 10 mg/ml pepstatin, aprotinin, and leupeptin, and 1 mM PMSF. The homogenates were centrifuged (15 min at 50,000 × g) and stored at -80°C. We measured the concentrations of hippocampal IL-1β and IL-1α by using homogeneous time-resolved fluorescence (HTRF) with mouse IL-1β (63ADK010PEB-JP) and mouse IL-1α (63ADK068PEB-JP) cytokine determination kits from Cisbio Japan (Tokyo, Japan). For the protein assay, we used a Coomassie (Bradford) Protein Assay Kit from Thermo Scientific (Rockford, IL, United States).
4
Transgenic Algae Protein Extraction
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Frozen pellets of sorted transgenic algae populations containing each construct were thawed, then resuspended and lysed in 1X BugBuster Protein Extraction Reagent with 0.06% Benzonase® endonuclease for 10 min on ice. Lysate was centrifuged at 5000 × g for 5 min at 4 °C to pellet cell debris, then supernatant was recovered as clarified lysate. Total soluble protein (TSP) concentration was measured using the Thermo Scientific™ Coomassie (Bradford) Protein Assay Kit as per the manufacturer’s instructions. Lysates were then normalized to 3 μg TSP and mixed with 4 × Laemmli buffer with 10% vol/vol β-mercaptoethanol. Samples were heated in an 80 °C-water bath for 10 min then allowed to cool. Proteins were separated by SDS-PAGE on 12% Mini-PROTEAN® TGX™ Precast Protein Gels at 200 V, then transferred onto nitrocellulose membrane at 15 V for 1 h. After blocking with TBSMT + 1% PVP-40 (Haycock 1993 (link)), membranes were probed with an anti-GFP monoclonal antibody conjugated to alkaline phosphatase (abcam, ab6661). The AccuRuler RGB Plus protein marker was used for reference band sizes, while wild-type C. reinhardtii strain CC125 was used as a negative control, and the high GFP-expressing single clone from the AR1 construct was used as a positive control.
5
Immunoblot Analysis of Cell Signaling
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Cells were lysed in RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, and protease inhibitors) and briefly sonicated. Protein content was measured using the Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of cell lysate were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare Biosciences, Foster City, CA, USA). Membranes were immunoblotted with antibodies against HMGB2 (Abcam, Cambridge, UK), p16, p21, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), and detected via chemiluminescence using ECL detection reagents.
6
Planarian Acetylcholinesterase Activity Assay
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After exposure, the planarians were washed 3X with IO water and then homogenized in 200 µL 1% Triton X-100 in PBS as described in (Hagstrom et al, 2017 (link); 2018 (link)). Briefly, after sitting on ice for about 15–20 min, the homogenate was centrifuged at 20,817 x g at 4°C for 30 min. The supernatant (clarified homogenate) was transferred to a clean, chilled tube and subsequently used. An Ellman assay (Ellman et al, 1961 (link)) was performed on the clarified homogenate using an Acetylcholinesterase Activity Assay kit (Sigma-Aldrich). Absorbance was read at 412 nm every minute for 10 min using a SpectraMax ABS Plus (Molecular Devices, San Jose, CA) spectrophotometer. AChE activity was calculated as the rate of change of absorbance per minute during the linear portion of the reaction. AChE activity was normalized by protein concentration as determined by a Coomassie (Bradford) protein assay kit (Thermo Scientific, Waltham, MA) and compared to the average respective solvent control samples (set at 100% activity) tested on the same day. Activity measurements were performed with 3 technical replicates per condition and 4 independent experiments (biological replicates).
7
Protein Quantification Using Proximity Extension Assay
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Sonicate fluids were thawed, and total protein concentrations of each determined by the Coomassie (Bradford) Protein Assay Kit (23,200, Thermo Scientific, Rockford, lL)52 (link). Samples were diluted to 700 µg/ml total protein concentration in 0.9% sodium chloride (2F7122, Baxter, Deerfield, IL) and 50 µl aliquots of each sample randomized into wells of twin.tec skirted 96 well PCR plates (951,020,460, Eppendorf, Hamburg, Germany). Samples were frozen on dry ice and sent to Olink Proteomics (Uppsala, Sweden), where protein quantification was performed using PEA with the Olink Proteomics - Inflammation Panel, including 92 target proteins (Supplemental Fig. 1 )35 (link). Olink Proteomics uses Normalized Protein eXpression (NPX), an arbitrary log2 value, for protein normalization and quantification.
8
Quantitative Tau Protein Analysis
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For biochemical analysis, half hippocampi were lysed in cold-lysis buffer (62.5 mM Tris hydrochloride, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 2.3% sodium dodecyl sulfate [SDS], 5 mM NaF, 100 µM Na3VO4, 1 mM EDTA, 1 mM EGTA) containing protease and phosphatase inhibitors (Roche España, Barcelona, Spain) and boiled at 100 °C61 (link). Protein content was quantified with the Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific, USA), resolved on SDS-polyacrylamide gel electrophoresis and detected by Western blotting using the following antibodies: rabbit anti-tau (Tau17025; 1:5000), mouse anti-phosphorylated Thr231 (AT180; 1:200), Ser202 (CP13; 1:250), and Ser396/404 (PHF-1; 1:250), abnormal tau conformation (aa 5–15 and 312–322; MC1), total tau TG5 (Tau 220–242; 1:500), and phosphorylated Ser9 GSK3β (1:1000; Cell Signaling, Danvers, Massachusetts) and anti-GSK3β (1:2500; BD Biosciences, San Jose, CA, USA), GFAP (1:250; Agilent, Santa Clara, CA, USA) and GAPDH (1:100000; Thermo Fisher Scientific). Bands were detected with enhanced chemiluminescent reagent in a ChemiDoc MP System (Bio-Rad) and quantified in a linear range using the ImageLab 5.2.1 software. Original blot images are shown in Supplementary Information.
9
Urinary Protein Biomarker Quantification
10
Soybean and N. benthamiana Protein Extraction and Co-IP
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Total proteins from soybean Forrest c.v. and N. benthamiana were extracted in lysis buffer containing 5mM DTT, 1% (v/v) NP40, 1mM sodium molybdate, 1 mm NaF, 1 mm PMSF, 1.5 mm Na3VO4, 100 mm NaCl, 2 mm EDTA, 50 mm Tris–HCl at pH 7.5, 10% (v/v) glycerol and one tablet from the plant protease and phosphatase inhibitors at 1:100 mL (Thermo Scientific). Protein concentration was quantified using Coomassie Bradford Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). For in planta co‐IP analysis, anti‐SNAP18 and anti‐SHMT08 polyclonal antibodies were immobilized in a column containing Aminolink Plus coupling resin (Pierce Co‐Immunoprecipitation Kit), and then, immunoblot analysis of root protein fraction samples from soybean Forrest, soybeans Essex (homologous system), or of leaf protein from N. benthamiana (heterologous system) was incubated overnight with the immobilized antibodies. After extensive washes, the immunoprecipitated associated proteins were eluted. The eluted fraction was then used for both Western blotting and mass spectrometry analysis.
For Western blotting analysis, anti‐SNAP18 (Rockland Immunochemicals, Limerick, PA), anti‐SHMT (Agrisera #AS05 075) or anti‐HA (Thermo Scientific #RB‐1438) polyclonal antibodies were used. Anti‐Rubisco (Bioss #6988R) polyclonal antibodies were used as a negative control. For native gel analysis, DTT and SDS agents were removed.
For Western blotting analysis, anti‐SNAP18 (Rockland Immunochemicals, Limerick, PA), anti‐SHMT (Agrisera #AS05 075) or anti‐HA (Thermo Scientific #RB‐1438) polyclonal antibodies were used. Anti‐Rubisco (Bioss #6988R) polyclonal antibodies were used as a negative control. For native gel analysis, DTT and SDS agents were removed.
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