Biotinylated proteins enriched with streptavidin beads were processed into peptides via on bead digestion and analyzed by LC-MS/MS according to the previously described methods20 (link),30 (link),32 (link). The samples were incubated for 15 min at 65 °C in 50 mM ammonium bicarbonate with Tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) to a final concentration of 1 mM. Proteins were then digested with 0.5 µg trypsin (Roche, Cat. No. 03708969001) and 0.1 µg LysC (Wako Chemicals, Catalog number 125-05061) overnight at 37 °C. A second digestion was then carried out using 0.5 µg trypsin and 0.05 µg LysC for 3 h at 37 °C. Iodoacetamide (IAM) was added to the digested peptides to a final concentration of 2.5 mM and the samples were incubated at 37 °C for 30 min in the dark. Peptides were separated from the streptavidin beads using a magnet and acidified by adding formic acid to a pH of ~2–3. Peptides were then desalted with 50 mg Sep-Pak C18 cartridges (Waters). Eluted peptides were dried using a SpeedVac and resuspended in water. Peptide amount was then estimated using the Pierce Quantitative Colorimetric Assay (Thermo Fisher Scientific, Catalog number 23275).
Pierce quantitative colorimetric assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce quantitative colorimetric assay is a laboratory tool used to measure the concentration of proteins in a sample. It provides a quantitative analysis of protein content through a colorimetric reaction. The assay is designed to be accurate, reliable, and easy to use in various research and analytical settings.
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Lab products found in correlation
Sep pak c18 cartridge, by Waters Corporation (2 mentions)
Lys c, by Fujifilm (1 mentions)
Trypsin, by Roche (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Vivacon 500, by Sartorius (1 mentions)
Tmt 10 plex isobaric labeling reagent, by Thermo Fisher Scientific (1 mentions)
Tmtpro, by Thermo Fisher Scientific (1 mentions)
Tmt 10 plex isobaric label reagents, by Thermo Fisher Scientific (1 mentions)
Tandem mass tag reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using pierce quantitative colorimetric assay
1
Biotinylated Protein Enrichment and Peptide Analysis
2
Biotinylated Protein Digestion and Analysis
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Biotinylated proteins enriched with streptavidin beads were enzymatically digested into polypeptides and analyzed by LC–MS/MS. After absorbing the thawed beads with a magnetic frame, 100 µL of 5 mm dithiothreitol (DTT) in 50 mm triethylammonium bicarbonate (TEAB) (Sigma, Cat. No. T7408) was added, and the mixture was treated at 56°C for 30 min with shaking on a rotator. The beads were then washed with 200 µL 50 mm TEAB for 1 min and incubated at RT with 100 µL of 20 mm iodoacetamide (IAM) in 50 mm TEAB for 30 min under dark conditions. The beads were then washed once with 50 mm TEAB buffer. Finally, 1 µg of trypsin (Thermo Fisher Scientific, Cat. No. 90058) dissolved in 50 mm TEAB buffer (pH 8.0 to 8.5) was added to the beads followed by digestion overnight at 37°C. Magnetic beads were then collected on a magnetic separation rack, and the supernatant was added onto prewetted 10,000 MWCOHY ultrafiltration products of VIVACON 500 (Sartorius Stedim Biotech GmbH, Cat. No. VN01H02) followed by centrifugation at 16,160 × g for more than 20 min until all the peptides were collected into the tubes. Peptides were then quantified using the Pierce Quantitative Colorimetric Assay (Thermo Fisher Scientific, Cat. No. 23275). Samples were analyzed by Thermo Q-Exactive high-resolution MS (Thermo Scientific, Waltham, MA, USA) to examine the quality of the peptides.
3
Peptide Quantification and Labeling
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Peptide quantification was determined by Pierce quantitative colorimetric assay (Thermo Scientific). For each sample, 100 μg of peptide was resuspended in 0.1 M triethylammonium bicarbonate (TEAB) and incubated with a tandem mass tag (TMT) 10-plex isobaric labeling reagent (0.8 mg Thermo Scientific). The ratio of TMT to substrate was 0.4 mg reagent to 0.1 mg peptide. The reaction was carried out for 1 h at room temperature and quenched using 5% (v/v) hydroxylamine for 15 min. Equal amounts of each sample were combined in a new tube and desalted using a C18 Tip.
4
Quantitative Proteomic Profiling of Dialysis
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Prior to tandem mass tag (TMT) labeling, peptide quantification was performed by the Pierce quantitative colorimetric assay (Thermo Scientific). According to the manufacturer’s instructions, 100 μg of peptide per sample was resuspended in 0.1 M triethylammonium bicarbonate (TEAB). Peptides (five channels per condition, healthy, and pre- and post-dialysis) were labeled with TMTpro (Thermo Scientific) for 1 h at room temperature. To quench the reaction, 5% hydroxylamine was added to each sample, and the resulting mixture was incubated at room temperature for 15 min. After labeling, equal amounts of each sample were combined in a new microtube and desalted using a C18 Sep-Pak cartridge (Waters).
5
Isobaric Labeling for Peptide Quantification
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Peptide quantification was determined by Pierce quantitative colorimetric assay (Thermo Fisher Scientific) and 100 μg of peptide per sample was resuspended in 0.1 M triethylammonium bicarbonate (TEAB) and incubated with the TMT 10-plex isobaric label reagents (0.8 mg Thermo Fisher Scientific). The ratio of TMT to substrate was 0.4 mg regent to 0.1 mg peptide. Reaction was carried out for 1 h at room temperature. To quench the reaction, 5% hydroxylamine was added to each sample and incubated for 15 min. Equal amounts of each sample were combined in a new tube and a speed vac was used to dry the labeled peptide sample. The labeled peptides were desalted using C18 Tips (Thermo Fisher Scientific).
6
Tandem Mass Tag Labeling for Peptide Quantification
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Digestions were acidified with trifluoracetic acid (TFA, 0.5% v/v) and desalted on Sep-Pak® Vac cartridges (WatersTM, Milford, Massachusetts, United States, WAT054955) with a wash of 1 ml 0.1% TFA followed by elution in 70% acetonitrile 0.1% formic acid (FA). Peptides were dried by speed vacuum and resuspended in 29 µL of 50 mM triethylammonium bicarbonate. Peptide concentrations were checked by Pierce Quantitative colorimetric assay (Thermo Fisher Scientific, Waltham, Massachusetts, United States, 23275). The same amount of peptide from each sample was then labeled for 2 hours at room temperature with 0.2 mg of Tandem Mass Tag reagent (Thermo Fisher Scientific, TMT™ Isobaric Label Reagent Set, 90309, lot no. VI307195B). Labeling reactions were quenched by adding 0.3% hydroxylamine (v/v) to the reaction mixtures at room temperature for 15 min. Labeled peptides were then mixed and dried by speed vacuum.
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