Following electrophoresis and staining, a complete gel strip of EE was cut and digested with 500 ng of sequencing grade trypsin (Promega, Wisconsin, USA) in 200 μL of ammonium bicarbonate solution as described elsewhere [34 (link)]. The selected EE and FE bands from other gels (egg and female extracts) were manually excised and digested with 100 ng of sequencing grade trypsin (Promega) in 100 μL of ammonium bicarbonate as described elsewhere [34 (link)]. Digestion was stopped with 1% trifluoracetic acid (TFA), and a double extraction with acetonitrile (ACN) was performed. The final peptide solutions were vacuum-dried and resuspended with 25 μL of 2% ACN and 0.1% TFA (pH 2.0) for the EE and 9 μL of 2% ACN and 0.1% TFA (pH 2.0) for the individual EE and FE bands as previously described [32 (link)].
Sequencing grade trypsin
Manufactured by Promega
Sourced in United States, Germany, United Kingdom, Italy, France, Spain, Switzerland
Sequencing grade trypsin is a proteolytic enzyme used to cleave peptide bonds in protein samples, primarily for use in protein sequencing applications. It is purified to ensure high-quality, consistent performance for analytical processes.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Merck Group (81 mentions)
Dithiothreitol (dtt), by Merck Group (64 mentions)
Ammonium bicarbonate, by Merck Group (43 mentions)
Formic acid, by Merck Group (43 mentions)
Formic acid, by Thermo Fisher Scientific (28 mentions)
Trypsin, by Promega (28 mentions)
Urea, by Merck Group (25 mentions)
Mascot, by Matrix Science (18 mentions)
Trifluoroacetic acid, by Merck Group (17 mentions)
C18 ziptip, by Merck Group (16 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (15 mentions)
Acetonitrile, by Merck Group (14 mentions)
Chymotrypsin, by Promega (14 mentions)
Hlb oasis spe cartridges, by Waters Corporation (14 mentions)
Rapigest, by Waters Corporation (13 mentions)
Pngase f, by New England Biolabs (13 mentions)
Acetonitrile, by Thermo Fisher Scientific (12 mentions)
Nanodrop, by Thermo Fisher Scientific (12 mentions)
Lys c, by Fujifilm (12 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (12 mentions)
899 protocols using sequencing grade trypsin
1
Protein Identification via Trypsin Digestion
2
CFTR Interactome Identification
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Following CFTR immunoprecipitation, complexes eluted in a solution containing DTT were loaded into filtering columns and washed exhaustively with urea (8 M) in HEPES buffer. After alkylation with iodoacetamide, proteins were incubated overnight with sequencing-grade trypsin (Promega, Madison, WI, USA). Peptides were desalted and analyzed by mass spectrometry. In the case of peptide pull-down, the eluted proteins were loaded into filtering columns and washed with 8 M urea in HEPES buffer. The sample incubated with DTT was used for reduction and then with iodoacetamide for alkylation. Proteins were incubated overnight with sequencing-grade trypsin (Promega). Peptides were subjected to mass spectrometry analysis.
3
PEAK3 Immunoprecipitation and Trypsin Digestion
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For immunoprecipitated PEAK3 from HEK293 cells, eluates were combined and 10 μL of 8 M urea, 250 mM Tris, 5 mM DTT (final concentration ~1.7 M urea, 50 mM Tris, and 1 mM DTT) was added to give a final total volume of ~45 μL. Samples were incubated at 60 °C for 15 min and allowed to cool to room temperature. IODO was added to a final concentration of 3 mM and the mixture was incubated at room temperature for 45 min in the dark. DTT was added to a final concentration of 3 mM before adding 1 μg of sequencing-grade trypsin (Promega) and incubating at 37 °C overnight. Samples were acidified to 1% trifluoroacetic acid (TFA, pH <2) with 20% TFA stock and incubated for 30 min before desalting on C18 stage tip.
For purified PEAK3/14-3-3 complexes, 5 ug of the purified protein complex was resuspended in 10 μL of 8 M urea, 50 mM Tris, 1 mM DTT. Samples were incubated at 60 °C for 15 min and allowed to cool to room temperature. IODO was added to a final concentration of 3 mM and the mixture was incubated at room temperature for 45 min in the dark. DTT was added to a final concentration of 3 mM and the sample was then diluted with 90 μL of 50 mM Tris before adding 0.1 μg of sequencing-grade trypsin (Promega) and incubating at 37 °C overnight. Samples were acidified to 1% trifluoroacetic acid (TFA, pH <2) with 20% TFA stock and incubated for 30 min before desalting on C18 stage tip.
For purified PEAK3/14-3-3 complexes, 5 ug of the purified protein complex was resuspended in 10 μL of 8 M urea, 50 mM Tris, 1 mM DTT. Samples were incubated at 60 °C for 15 min and allowed to cool to room temperature. IODO was added to a final concentration of 3 mM and the mixture was incubated at room temperature for 45 min in the dark. DTT was added to a final concentration of 3 mM and the sample was then diluted with 90 μL of 50 mM Tris before adding 0.1 μg of sequencing-grade trypsin (Promega) and incubating at 37 °C overnight. Samples were acidified to 1% trifluoroacetic acid (TFA, pH <2) with 20% TFA stock and incubated for 30 min before desalting on C18 stage tip.
4
Spermatozoa Proteome Sample Preparation
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The collected human spermatozoa samples were denatured individually in 8 M urea/1 M NH4HCO3 and went through ultrasonication on ice by Ultrasonic Cell Distribution System. Thereafter, denatured samples were centrifuged at 15,000g for 20 min, and supernatant was collected for protein concentration measurement by BCA reagent (Beyotime). Spermatozoa proteins (1 mg) were reduced by 5 mM dithiothreitol (DTT) at 37 °C for 1 h with gentle shaking, then alkylated by 15 mM iodoacetamide at room temperature (RT) for 30 min in the dark. Subsequently, another 2.5 mM DTT was added and incubated for 10 min at RT. Protein samples were diluted twofold with deionized water and digested by sequencing grade trypsin (protein: enzyme, 100:1, w/w; Promega) at 37 °C for 2 h with gentle shaking as the first digestion. Afterward, samples were diluted fourfold with deionized water and sequencing grade trypsin (protein: enzyme, 100:1, w/w; Promega) was used to digest proteins into peptides again by incubation at 37 °C with gentle shaking overnight, namely the second digestion. The samples were acidified with trifluoroacetic acid (TFA) and centrifuged at 15,000g for 15 min to remove any particulate matter. The digested peptides were desalted with C18 column (Waters) and eluted with 50% acetonitrile (ACN)/0.1% TFA. The peptide concentration was measured by BCA reagent (Beyotime).
5
Protein Extraction and Trypsin Digestion
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Following electrophoresis and staining, a complete gel strip of egg extract was cut and digested with 500 ng of sequencing grade trypsin (Promega, Wisconsin, USA) in 200 µL of ammonium bicarbonate solution as described elsewhere [30] . The selected bands from other gels (egg and female extracts) were manually excised and digested with 100 ng of sequencing grade trypsin (Promega) in 100 µL of ammonium bicarbonate as described elsewhere [30] . Digestion was stopped with 1% tri uoracetic acid (TFA) and a double extraction with acetonitrile (ACN) was performed. The nal peptide solution was vacuum-dried and resuspended with 25 µL of 2% ACN and 0.1% TFA (pH 2.0) for the EE and 9 µL of 2% ACN and 0.1% TFA (pH 2.0) for the individual bands as previously described [28] .
6
SDS-PAGE Protein Identification Protocol
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Proteins were separated through 1D-SDS-PAGE on 10% of polyacrylamide gels. Next, gels were stained with 0.1% of Coomassie R-250. Selected bands were excised from gels and sent to the Center for Chemical and Biological Studies by Mass Spectrometry (CEQUIBIEM), Faculty of Exact and Natural Sciences, University of Buenos Aires, where protein identification analysis was performed. Briefly, bands were de-stained with 50 mM of ammonium bicarbonate/acetonitrile (50/50% v/v), reduced with DTT, followed by alkylation with iodoacetamide. Trypsin sequencing grade was used for in-gel digestion (Promega, Madison, WI, USA). The tryptic digested peptides were resuspended in 0.1% formic acid in water, injected into Easy nLC 1000 (Thermo Scientific), and analyzed by tandem mass spectrometry using a QExactive Orbitrap mass spectrometer (Thermo Scientific) [61 (link)].
7
Protein Purification and Digestion
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Protein solution containing SDS and DTT were loaded into filtering columns and washed exhaustively with 8M urea in HEPES buffer26 (link). Proteins were reduced with DTT and alkylated with IAA. Protein digestion was performed by overnight digestion with trypsin sequencing grade (Promega).
8
Porcine Peptide Standard Preparation
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Trypsin (sequencing grade) was obtained from Promega (Fitchburg, WI, USA). Formic acid, acetonitrile (Ultra-performance liquid chromatography [UPLC]-grade), were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ultra high purity water was prepared using a Milli-Q water purification system (Millipore Corporation, Bedford, MA). Syringe filters (0.22 μm) were purchased from Millipore (Billerica, MA, USA). All other chemicals and reagents were of the highest grade available. Porcine marker peptide standards were synthesized by Jill Biochemical Co., Ltd. (Shanghai, People's Republic of China). Gelatin samples were prepared in the laboratory.
9
Proteomics Profiling with Standard Reagents
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Conalbumin was purchased from Fluka. Ribonuclease B, beta casein, fibrinogen, and myoglobin were purchased from SIGMA AltrichSt. Louis, MO. 6 Bovine Tryptic Digest Equal Molar Mix was purchased from Bruker, Billerica, MA. The HEK-293cells were kindly provided by Dr. Audrey van Drogen (ETH, Zurich). Iodoacetamide, tris(2-carboxyethyl)phosphine, trifluoroacetic acid, acetonitrile (ACN), HPLC water, ammonium bicarbonate, acetaminophen, and urea were purchased from SIGMA-Aldrich. Trypsin sequencing grade was purchased from Promega, Madison, WI. RapiGest was purchased from Waters, Milford, MA. Synthetic, heavy-labeled peptides were purchased from Thermo Scientific, Waltham, MA.
10
Quantitative Proteomics of Subcellular Fractions
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Protein concentrations of inner and OM fractions were determined by Pierce™ bicinchoninic acid (BCA) Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). For liquid chromatography-mass spectrometry (LC/MS) analysis, all samples were adjusted to the protein concentration of 0.4 mg mL−1 in 10 mmol L−1 HEPES of pH 7.4. The samples were digested with 10 μg trypsin (sequencing grade) (Promega, Madison, WI, USA) overnight at 37 °C. The digestion was stopped by adding 5 μL 50% formic acid and the generated peptides were purified using a ZipTip C18 (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions and dried using a Speed Vac concentrator Plus (Eppendorf, Hamburg, Germany). The tryptic peptides were analyzed using an Ultimate 3000 nano-UHPLC system connected to a Q Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) equipped with a nano electrospray ion source.
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