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.
Tpck trypsin 20233
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.
Lab products found in correlation
8 protocols using tpck trypsin 20233
Proteome Analysis by FASP Trypsin Digestion
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.
Phosphoproteome Enrichment and Analysis
Secretome Protein Extraction from Mesenchymal Stem Cells
Protein Preparation and Trypsin Digestion
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.
Phage Proteome Profiling by Mass Spectrometry
Phage Particle Proteomic Analysis by FASP Protocol
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.
Trypsin Digestion for Mass Spectrometry
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.
Trypsin Digestion and Mass Spectrometry Analysis
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