2×106 T cells were incubated for 45min in ice-cold lysis buffer (10mM Tris, 50mM NaCl, 5mM EDTA and 1% Triton X-100) containing various protease and phosphatase inhibitors (50mM NaF, 1mM Na3VO4, 30mM sodium pyrophosphate, 1mM PMSF, 2µg/ml leupeptin, 2µg/ml aprotinin). Lysates were then centrifuged at 12.000 g for 15min at 4°C to remove insoluble material. Protein concentration was determined using the Coomassie protein assay reagent (Sigma Aldrich). Proteins (20µg per lane) were separated in 4 to12% gradient (wt/vol) Bis-Tris gels (Life Technologies) and transferred to nitrocellulose (Life Technologies) or PVDF (Millipore) membrane. Membranes were blocked for 1h with Tris-buffered saline solution containing 0.05% Tween (TBS-T) and 5% non-fat dry milk at room temperature. Following blocking, membranes were incubated overnight at 4°C with the indicated antibody in blocking solution on a shaking surface and were then washed and incubated with the appropriate secondary HRP-conjugated antibody for 1.5h at room temperature. Detection was performed with the Clarity ECL Western Blotting detection reagents (Biorad) and membranes were visualized by the ChemiDoc XRS+ Molecular Imager (Biorad). Densitometric analysis was performed using the ImageJ software and results are expressed as densitometric ratios of tyrosine phosphorylated proteins or SAP over β-actin ± SEM.
Coomassie protein assay reagent
Manufactured by Merck Group
Sourced in United States
Coomassie protein assay reagent is a laboratory product used for quantifying total protein concentrations in biological samples. It is a colorimetric assay that measures the binding of the Coomassie dye to proteins, resulting in a color change that is proportional to the protein concentration. The reagent provides a simple and reliable method for determining protein levels in a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence reagent, by Bio-Rad (2 mentions)
Autoradiography film, by Genesee Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Ripa cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose, by Thermo Fisher Scientific (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Clarity ecl western blotting detection reagent, by Bio-Rad (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
P21waf cip1, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
P16ink4a, by Abcam (1 mentions)
4mu neuac, by Merck Group (1 mentions)
Mild stripping buffer, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
7 protocols using coomassie protein assay reagent
1
Protein extraction and Western blotting
2
Citrate Synthase Purification from Porcine Heart
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Citrate Synthase from Porcine Heart ammonium sulfate suspension (C3260) was purchased from Sigma Aldrich. Suspension was centrifugated at 14,000 rpm for 10 min at 4°C. Supernatant was discarded and the pellet was resuspended in 750 μl MilliQ water. To remove residual ammonium sulfate, citrate synthase was desalted in MilliQ water using a 5 ml HiTrap Desalting Column (GE Healthcare). Fractions were evaluated for protein content via staining 30 μl of each fraction with 100 μl of Coomassie protein assay reagent (Sigma) diluted 1:5 in MilliQ water. Fractions with detectable protein content were pooled and concentrated using an Amicon Ultra 0.5 ml 10K centrifugal filter unit (Merck Millipore Ltd). Final concentration determined via measuring absorbance at 280 nm on a Nanodrop 2000 spectrophotometer (Thermo Scientific).
3
Age-related RPE Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from RPE/eyecup of mice (2, 12 and 18 months old) or ARPE-19 cells or primary RPE cells using RIPA cell lysis buffer (Thermo Scientific) containing protease and phosphatase inhibitors and concentration was determined using the coomassie protein assay reagent (Sigma-Aldrich, USA). Equivalent amount of protein samples were subjected to SDS–PAGE, transferred to PVDF membranes, and then incubated with primary antibodies: SIRT-1, p21Waf/Cip1 (1:1000; Cell signaling) and p16INK4a (1:500; Abcam) overnight at 4°C. Next day, blots were washed with TBST and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Sigma-Aldrich, USA) for 60 min with gentle shaking at room temperature. Blots were then washed (with TBST) and developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). β-actin (1:3000; Sigma-Aldrich, USA) expression was evaluated to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
4
Western Blot Analysis of Redox Regulators in Mouse and Human RPE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from RPE/eyecup of mice or from human RPE (ARPE-19) cells using RIPA cell lysis buffer (Thermo Scientific, USA) containing protease and phosphatase inhibitors and concentration was determined using the Coomassie protein assay reagent (Sigma-Aldrich, USA). Equivalent amount of protein samples (40–60 μg) were subjected to SDS–PAGE, transferred onto PVDF membranes, and then incubated with primary antibodies: Nrf2 (1:250, cell signaling), GCLC, HO-1 (1:1000, Abcam, USA), NQO1 (1:1000, Santa cruz biotech, USA) overnight at 4 °C. Next day, blots were washed with TBST (tris buffered saline with tween 20) and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Sigma-Aldrich, USA) for 60 min with gentle shaking at room temperature. Blots were then washed with TBST and developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). β-actin (1:3000; Sigma-Aldrich, USA) expression was evaluated to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ) and expressed as fold change.
5
Enzymatic Activity Assay for NEU1 Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The enzymatic activity in total cell lysates was determined as previously described [1] (link) using 1 mM 4MU-NeuAc (4-methylumbelliferyl-N-acetyl-α-d -neuraminic acid, Sigma) as substrate. Assays were performed in triplicate with 10 µl sample volume in a final volume of 30 µl. Samples were incubated at 37°C for 30 min. Reactions were stopped using 0.2 M Glycine/NaOH pH 10.8 and activity was measured using Jasco FP-770 Spectrofluorimeter. Fluorescent intensity was referred to a standard concentration curve of 4MUB (4-methylumbelliferone). Protein concentration was determined by dye-binding assay (Coomassie Protein Assay Reagent, SIGMA) according to manufacturer's manual. A standard two-tailed t-test was calculated for every mutant taking into account all the replicated experiments to assess the significance of enzymatic activity variation compared to the wild-type NEU1. When calculating the enzymatic activity of V217A and D234N mutant proteins, 3 samples with significant lower level of normalized PPCA protein (determined as described above) were discarded resulting in a final dataset of 5 biological replicates for each mutant.
6
Western Blot Analysis of Protein Samples
7
Pollen Protein Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pollen samples (10 mg) were suspended in Eppendorf tubes with 12 mg of zirconia beads (0.5 nm), 200 µL of phosphate buffered saline (1:20 w/v) at pH 7.4 and 10 µL of protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Then, each tube was stirred using a Mini-Beadbeater TM at 4 • C (3 cycles each with the duration of 30 s with pause intervals of 15 s at 16,100 g). Subsequently, the samples were kept at 4 • C under constant (orbital) stirring for 2 h, centrifuged twice (16,100 g for 20 min at 4 • C) and the supernatant was filtered (0.45 µm Millipore filter) and centrifuged again in the conditions previously described.
The soluble protein content of all pollen extracts was quantified through a colorimetrical reaction with the Coomassie Protein Assay Reagent (sigma-Aldrich) in microtiter plates (300 µL/well at 595 nm) according to the Bradford method [38] (link). Different BSA concentrations (0-2000 µg/mL) were used to estimate a standard curve for protein calibration.
The soluble protein content of all pollen extracts was quantified through a colorimetrical reaction with the Coomassie Protein Assay Reagent (sigma-Aldrich) in microtiter plates (300 µL/well at 595 nm) according to the Bradford method [38] (link). Different BSA concentrations (0-2000 µg/mL) were used to estimate a standard curve for protein calibration.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!