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Tpck trypsin 20233

Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania

TPCK Trypsin 20233 is a laboratory reagent used for the digestion and cleavage of proteins. It is a proteolytic enzyme derived from bovine pancreas and is commonly used in cell culture and biochemical applications.

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8 protocols using tpck trypsin 20233

1

Proteome Analysis by FASP Trypsin Digestion

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Cells were grown for 24 h in RH1-free growth media, rinsed 3 times with PBS (37°C), and lysed with 0.5 ml of urea/thiourea lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Vilnius, Lithuania). The lysates were sonicated for 1 min at the amplitude of 20% and 0.4 s pulsations on/off cycles (Sonopuls HD 2070, Bandelin, Berlin, Germany). Homogenized lysates were centrifuged at 20,000 g for 15 min at 4 °C, and the supernatants were collected. The lysates then were stored at −86 °C.
Trypsin digestion was done according to a modified filter-aided sample preparation (FASP) protocol as described by Wisniewski et al. [99 (link)]. Briefly, proteins were diluted in 8 M urea, and loaded on Amicon Ultra-30 kDa filters. Following two washes with urea, proteins were alkylated with 50 mM iodoacetamide. Filters were washed twice with urea and twice with 50 mM NH4HCO3. Proteins digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Vilnius, Lithuania). After overnight digestion, peptides were recovered by centrifugation and then two additional washes using 50% CH3CN were combined, acidified, lyophilized, redissolved in 0.1% formic acid and then fractionated off-line.
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2

Phosphoproteome Enrichment and Analysis

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The Pro-Q Diamond Phosphoprotein Enrichment kit/reagents and NuPAGE precast 4%–12% Bis–Tris polyacrylamide gels were purchased from Life Technologies. Bradford Protein concentration assay kit and TPCK Trypsin 20233 for protein digestion were obtained from Thermo Fisher. Protease and phosphatase (for serine, threonine, and tyrosine) inhibitors and all other reagents were of analytical grade and quality and were obtained from Sigma (if not stated otherwise).
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3

Secretome Protein Extraction from Mesenchymal Stem Cells

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MenSCs’ culture medium is aspirated and collected in 5 mL tubes and centrifuged for 10 min at 2000× g to remove cells and cell debris. After centrifugation, the supernatant is collected and filtered through a 0.22 μm filter. After filtration, the samples are cleaned and concentrated using ProteoMinerTM Sequential Elution Large-Capacity kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The secretome protein concentration was measured using the Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The secretome proteins were trypsinized according FASP protocol, as described by Wisniewski et al. [32 (link)]. Briefly, proteins were diluted in 8 M urea; following two washes with urea, proteins were alkylated with 50 mM iodoacetamide (GE Healthcare Life Sciences, MA, USA). Protein concentrators were washed twice with urea and twice with 50 mM NH4HCO3. Proteins were digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Vilnius, Lithuania). After overnight digestion, peptides were collected from the concentrators by centrifugation at 14,000× g for 10 min and additionally eluted using 20% CH3CN. The eluates were combined, acidified with 10% CF3COOH and lyophilized in vacuum centrifuge. The lyophilized peptides were redissolved in 0.1% formic acid.
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4

Protein Preparation and Trypsin Digestion

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Samples of proteins excised from gels were prepared as described by Shevchenko et al. [28 (link)]
Whole proteome samples were digested with trypsin according to FASP protocol as described by Wisniewski et al. [29 (link)] Briefly, samples were diluted in 8 M urea following two washes with urea and alkylated with 50 mM of IOA (GE Healthcare Life Sciences, Marlborough, MA, USA). Protein concentrators were washed twice with urea and twice with 50 mM of ammonium bicarbonate. Proteins were digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Waltham, MA, USA). After overnight digestion, peptides were collected from the concentrators by centrifugation at 14,000× g for 10 min and additionally eluted using 20% acetonitrile. The eluates were mixed, acidified with 10% trifluoracetic acid and lyophilized in a vacuum centrifuge. The lyophilized peptides were redissolved in 0.1% formic acid.
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5

Phage Proteome Profiling by Mass Spectrometry

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CsCl-purified phage particles were concentrated on Amicon Ultra-0.5 mL 30 kDa centrifugal filter unit and were denatured in 8 M urea, 100 mM DTT solution with continuous rotation at 800 rpm in the temperature controlled shaker for 3 hours at 37 °C. Trypsin digestion was performed according to a modified FASP protocol [46 (link),47 (link)]. Briefly, phage particles were washed with buffer containing 8 M urea. The proteins were alkylated using iodoacetamide. Buffer was exchanged by washing twice with 50 mM NH4HCO3, and proteins digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Rockford, IL, USA). Then the peptides were recovered by centrifugation and washed twice with 50% CH3CN. Afterwards, the samples were combined, acidified, lyophilized, redissolved in 0.1% formic acid and analyzed by mass spectrometry.
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6

Phage Particle Proteomic Analysis by FASP Protocol

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CsCl-purified phage particles were concentrated on Amicon Ultra-0.5 mL 30 kDa centrifugal filter unit and were denatured in 8 M urea, 100 mM DTT solution with continuous rotation at 800 rpm in the temperature controlled shaker for 3 hours at 37°C.
Trypsin digestion was performed according to a modified FASP protocol as described by Wisniewski et al. [41] (link). Briefly, phage particles were washed with buffer containing 8 M urea. The proteins were alkylated using iodoacetamide. Buffer was exchanged by washing twice with 50 mM NH4HCO3, and proteins digested overnight with TPCK Trypsin 20233 (Thermo Scientific, USA). Then peptides were recovered by centrifugation and washed twice with 50% CH3CN. Afterwards, samples were combined, acidified, lyophilized, redissolved in 0.1% formic acid and analysed by mass spectrometry.
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7

Trypsin Digestion for Mass Spectrometry

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Samples were concentrated on Amicon ultra-0.5 mL 30 kDa centrifugal filter unit and were denatured in 8 M urea, 100 mM DTT solution with continuous rotation at 800 rpm in the temperature controlled shaker for 3 hours at 37°C.
Trypsin digestion was done according to a modified FASP protocol as described by Wiśniewski et al. [23 (link)]. Briefly, samples were washed with buffer containing 8 M urea. The proteins were alkylated using iodoacetamide. Buffer was exchanged by washing two times with 50 mM NH4HCO3 and proteins digested overnight with TPCK Trypsin 20233 (Thermo Scientific, USA). After overnight digestion, peptides were recovered by centrifugation and then two additional washes using 50% CH3CN were combined, acidified, lyophilized, redissolved in 0.1% formic acid, and then analysed by mass spectrometry.
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8

Trypsin Digestion and Mass Spectrometry Analysis

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Proteins were concentrated on Amicon Ultra-0.5 mL 30 kDa centrifugal filter. Trypsin digestion was performed according to a modified FASP protocol as described by Wisniewski et al. [11] . Briefly, proteins were washed with buffer containing 8 M urea. The proteins were alkylated using iodoacetamide. Buffer was exchanged by washing twice with 50 mM NH 4 HCO 3 , and proteins were digested overnight with TPCK Trypsin 20233 (Thermo Scientific, USA). Then, peptides were recovered by centrifugation and washed with 20% CH 3 CN. Afterward, samples were lyophilized, redissolved in 0.1% formic acid, and analyzed by mass spectrometry (MS).
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