Single colonies (diameter >2 mm) were picked using a micropipette tip or a 10 μL loop, and resuspended in 200 μL of 50 mM ammonium bicarbonate (MS grade; Merck, Germany), sonicated for 1 min (3 s of sonication, 6 s of rest), centrifuged at 12,000 × g for 2 min and heated at 95°C for 5 min, after which the buffer was removed using 10K Nanosep centrifugal device with Omega membrane (Pall Corporation, Port Washington, NY, United States). Ammonium bicarbonate buffer (50 mM) was added along with sequencing grade trypsin (Promega Corporation, Madison, WI, United States), and the solution was microwaved in a water bath followed by heat treatment at 55°C. The peptide concentration was measured using the Pierce™ Quantitative Colorimetric Peptide Assay kit (Thermo Fisher Scientific, Waltham, MA, United States).
Pierce quantitative colorimetric peptide assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Spain
The Pierce™ Quantitative Colorimetric Peptide Assay kit is a laboratory product designed for the quantitative analysis of peptide concentrations. It utilizes a colorimetric reaction to measure the amount of peptide present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Fujifilm (2 mentions)
Lys c, by Fujifilm (2 mentions)
Gl tips sdb, by GL Sciences (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (2 mentions)
Iodoacetamide, by Fujifilm (2 mentions)
Trypsin, by Promega (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Cellulose membrane, by Merck Group (2 mentions)
Omega membrane, by Pall Corporation (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Tmt 10plex isobaric label reagent sets, by Thermo Fisher Scientific (1 mentions)
C18 sep pack cartridges, by Waters Corporation (1 mentions)
Ds 11 spectrophotometer, by DeNovix (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Human plasma, by Merck Group (1 mentions)
Potassium phosphate dibasic and potassium phosphate monobasic, by Merck Group (1 mentions)
Poloxamer 188 pluronic f 68, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
12 protocols using pierce quantitative colorimetric peptide assay kit
1
Protein Extraction and Trypsin Digestion
2
Quantitative Proteomic Profiling of Macrophages
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Digested proteins were resuspended in TEAB (50 mmol/liter). Peptide concentrations were determined using a Pierce™ Quantitative Colorimetric Peptide Assay kit (ThermoFisher Scientific) and calculated against a standard curve of known peptide concentrations with a plate reader set at an absorbance of 490 nm. 100 ug of each sample was taken, made up to 100 ug/ul in TEAB (50 mM) and labeled using TMT10plex™ Isobaric Label Reagent Sets (ThermoFisher Scientific) following the manufacturers protocol. TMT labels were resuspended in acetonitrile, added to samples and incubated at room temperature for 1 hour. Hydroxylamine (5%) was added for 15 minutes to quench the labeling reactions. The experimental design required 16 samples (4 each of untreated Gchfl/fl, untreated Gchfl/flTie2cre, MLPS/IFNγ Gchfl/fl and MLPS/IFNγ Gchfl/flTie2cre) which were therefore split over 2x TMT10plex™ kits (8 samples in each), with 2 labels used to link the sample sets and formed of identical pools containing equal quantities of all samples. Samples labeled using Kit 1 and Kit 2 were pooled separately and desalted using C18 Sep-Pack cartridges (Waters), vacuum dried and stored at −80°C.
3
Pancreatic Organoid EV Proteome Profiling
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Pancreatic organoid EV samples were pooled from fractions 1 and 2 after SEC isolation then dried in a speedvac. Samples were digested using PreOmics iST-NHS Kit 12 × (Product# PO00026) following the manufacturer’s instructions. Following digestion, all samples were taken to dryness by vacuum centrifugation, dissolved in 50 µl each of HPLC water and peptide concentrations determined using the Pierce Quantitative Colorimetric Peptide Assay Kit (Thermo Scientific). Total peptide recovery for EV samples in the 4 × 4 study was as follows: PDAC-1 (3.9 µg), PDAC-2 (6.3 µg), PDAC-3 (5.7 µg), PDAC-4 (4.3 µg), HC-1 (3.8 µg), HC-2 (1.5 µg), HC-3 (4.4 µg), HC-4 (3.5 µg). Samples PDAC-5 through PDAC-10 were processed separately and total peptide yields ranged from 5 to 20 µg.
4
Peptide Concentration Determination
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Concentrations were obtained by measuring absorbance at 280 nm using a DS-11+ spectrophotometer (DeNovix) and molar extinction coefficients calculated from the amino acid composition (ExPASy/ProtParam program) (100 ). ε0 (CaM, Cav1.2-IQ1665-1685) = 2980 M−1 cm−1; ε0 (Cav1.2-NSCaTE51-67, RyR23581-3608) = 5500 M−1 cm−1.
For CaMKIIδ294-315 and Syntide-2 peptides, because the amino acid sequence does not contain any tryptophan or tyrosine, concentrations were determined using the Pierce Quantitative Colorimetric Peptide Assay kit (Thermo Fisher Scientific) as per the manufacturer’s instruction.
For CaMKIIδ294-315 and Syntide-2 peptides, because the amino acid sequence does not contain any tryptophan or tyrosine, concentrations were determined using the Pierce Quantitative Colorimetric Peptide Assay kit (Thermo Fisher Scientific) as per the manufacturer’s instruction.
5
Tandem Mass Tag Peptide Labeling
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Peptides were desalted on a 50-mg Sep-Pak Vac (Waters Corporation) using a vacuum manifold. After elution from the column in 70% acetonitrile (ACN) and 0.1% formic acid (FA), peptides were dried by speed vacuum and resuspended in 24 μl of 50 mM triethylammonium bicarbonate. Peptide concentration was measured using the Pierce Quantitative Colorimetric Peptide Assay Kit (23275, Thermo Fisher Scientific) to ensure that an equal amount of each sample was labeled. Samples were then tandem mass tag (TMT)–labeled with 0.2 and 2 mg of reagent for global and phosphoproteomic studies, respectively, for 2 hours at room temperature (global: TMT lot UD278759A: PyMT ambient-1 126, PyMT physioxia-1 127C, PyMT ambient-2 128C, PyMT physioxia-2 129C, Her2 ambient-1 127N, Her2 ambient-2 128N, Her2 physioxia-1 129N, and Her2 physioxia-2 130N; phospho: TMTpro lot UL297970: PyMT ambient 126N and PyMT physioxia 128N). Labeling reactions were quenched with hydroxylamine at room temperature for 15 min. Labeled peptides were then mixed by global and phospho sets, respectively, and dried by speed vacuum.
6
Docetaxel-Loaded Polymeric Nanoparticles
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2.1 Materials Sodium Chloride, DAPI, poloxamer 188 (Pluronic F68) and methanol (MeOH) were from Sigma-Aldrich, Milan, Italy. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and the Pierce™ Quantitative Colorimetric Peptide Assay kit were from Thermo Fisher Scientific, Monza, Italy. Dichloromethane (DCM) and acetone were from Honeywells, Monza, Italy. Docetaxel (DTX) was provided by Alpha Aesar. Potassium phosphate dibasic and potassium phosphate monobasic, sodium azide, potassium chloride, sodium phosphate dibasic, human serum albumin (HSA) and human plasma were used as received (Sigma-Aldrich, Italy). Dialysis membranes (MWCO 3500, regenerated cellulose) were used as received (Spectrapor, Italy). The aminoterminated H2N-PCL4.7K-NH2 (daPCL) was synthesized and characterized as previously reported (Conte et al., 2019a) .
7
Secretome Analysis of Conditioned Media
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Secretome analysis of CM was conducted according to a previously reported protocol.19 (link),22 (link) Briefly, M0-CM and M2-CM were concentrated using Amicon Ultra 3 K filters (MilliporeSigma, Burlington, MA, USA) and precipitated by methanol/chloroform. The resulting pellets were resolved with MS buffer (8 M urea and 50 mM Tris-HCl pH 8.0), reduced with 5 mM DTT (Fujifilm Wako) for 30 minutes at room temperature (24-26 °C), and alkylated with 27.5 mM iodoacetamide (Fujifilm Wako) for 30 minutes in the dark at room temperature. After 8-fold dilution with 50 mM Tris-HCl pH 8.0, proteins were digested with 50 ng Lys-C (Fujifilm Wako) and trypsin (Promega, Madison, WI, USA) overnight at 37 °C. Peptides were purified using GL-Tips SDB (GL Sciences, Tokyo, Japan) according to the manufacturer’s protocol. Peptide concentrations were determined using a Pierce quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). Then, peptides (300 ng each) were injected into an EASY-nLC1200 system connected to an Orbitrap Fusion (Thermo Fisher Scientific) MS system. The scan range was m/z 350-1500. Protein identification and label-free quantification were performed using Proteome Discoverer 2.4 (Thermo Fisher Scientific).
8
Synaptic Peptide Extraction and Quantification
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Peptides were
extracted from synaptosomes by bringing the samples to 20 mM HCl (pH
3), incubation on ice for 15 min, centrifugation (14,000g for 30 min), and collection of the supernatant containing peptides.
This peptide extract was then filtered through a 10 kDa molecular
weight cutoff (MWCO) membrane (Millipore, Burlington, CA) by centrifugation
(14,000g for 60 min), including rinsing the membrane
with 0.5 M NaCl and 10 mM HCl with a second centrifugation. Filtrates
from the two centrifugation steps through the 10 kDa MWCO membrane
were combined and neutralized with 1 M ammonium bicarbonate to ∼30
mM. Peptide concentration was determined using a Pierce Quantitative
Colorimetric Peptide Assay Kit (Thermo Fisher).
extracted from synaptosomes by bringing the samples to 20 mM HCl (pH
3), incubation on ice for 15 min, centrifugation (14,000g for 30 min), and collection of the supernatant containing peptides.
This peptide extract was then filtered through a 10 kDa molecular
weight cutoff (MWCO) membrane (Millipore, Burlington, CA) by centrifugation
(14,000g for 60 min), including rinsing the membrane
with 0.5 M NaCl and 10 mM HCl with a second centrifugation. Filtrates
from the two centrifugation steps through the 10 kDa MWCO membrane
were combined and neutralized with 1 M ammonium bicarbonate to ∼30
mM. Peptide concentration was determined using a Pierce Quantitative
Colorimetric Peptide Assay Kit (Thermo Fisher).
9
Secretome Analysis of Conditioned Media
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Secretome analysis of CM was performed based on a previously reported protocol58 (link). In brief, SHED-CM and Fibro-CM were concentrated using an Amicon Ultra 3 K filter (Millipore). Methanol/chloroform precipitation was performed for protein purification. Resulting pellets were resolved by MS buffer (8 M urea and 50 mM Tris–HCl, pH 8.0), followed by reduction in 5 mM DTT (Fujifilm Wako) and alkylation in 27.5 mM iodoacetamide (Fujifilm Wako) for 30 min in the dark at room temperature. After being diluted eight times in 50 mM Tris–HCl (pH 8.0), proteins were digested overnight with 50 ng of Lys-C (Fujifilm Wako) and trypsin (Promega, Madison, WI, USA) at 37 °C. Peptides were purified using GL-Tips SDB (GL Science, Tokyo, Japan) based on the manufacturer's protocol. The concentration of peptides was measured using a Pierce quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). Subsequently, peptides (268 ng each) were injected into an EASY-nL connected to a Q-Exactive PLUS (Thermo Fisher Scientific) mass spectrometry system. Protein identification and label-free quantification were performed using Proteome Discoverer 2.2 (Thermo Fisher Scientific).
10
Peptide Concentration Determination
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The sample was dissolved in 50 μL of distilled water at a dilution ratio of 1:10, and the concentration was determined using the Pierce Quantitative Colorimetric Peptide Assay kit (Thermo Fisher Scientific).
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