One hundred micrograms of the protein sample was mixed with dithiothreitol solution at a final concentration of 100 mM and then incubated for 5 min at 95°C. Two hundred mL of 8 M urea and 150 mM Tris-HCl, pH 8.0 were added after the samples had cooled to room temperature. The mixtures were transferred to an ultrafiltration filter (10-kDa cutoff, Sartorius, Germany) and centrifuged at 14 000 × g for 15 min. Subsequently, 100 μL of iodoacetamide solution (50 mM iodoacetamide in 8 M urea and 150 mM Tris-HCl, pH 8.0) was added and incubated for 30 min at room temperature in the dark. Then 100 μL dissolution buffer (Applied Biosystems, Foster City, CA, USA) was added, mixed and centrifuged at 14 000 × g for 10 min and this step was repeated twice. Finally, 20 μL trypsin solution (2 μg trypsin in 20 μL dissolution buffer) was added, mixed and incubated for 16–18 h at 37°C. The digested peptides were collected into a new tube by centrifugation at 14 000 × g for 10 min.
Dissolution buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dissolution buffer is a solution used to facilitate the dissolution of solid or semi-solid substances in pharmaceutical and analytical applications. It provides a controlled environment to ensure the release and measurement of the active ingredients from a dosage form. The specific composition and properties of the buffer can vary depending on the requirements of the application.
Automatically generated - may contain errors
Lab products found in correlation
Ultrafiltration filter, by Sartorius (3 mentions)
Trypsin, by Promega (2 mentions)
Itraq reagent, by Thermo Fisher Scientific (1 mentions)
Polysulfoethyl a column, by Nest Group (1 mentions)
20ad high performance liquid chromatography system, by Shimadzu (1 mentions)
Sep pak vac c18, by Waters Corporation (1 mentions)
4 protocols using dissolution buffer
1
Protein Digestion for Mass Spectrometry
2
Quantitative Proteomic Analysis of Serum Samples
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iTRAQ reagents were purchased from Applied Biosystems (Foster City, CA, USA). Fourteen interfering highly abundant proteins from serum samples were removed using Agilent multiple affinity removal liquid chromatography (LC) column-Human 14 (MARS) (Shimadzu, Kyoto, Japan). Next, 100 μg of each extract was precipitated using acetone at −20°C and suspended in 20 μL Dissolution Buffer (Applied Biosystems). After reduction and alkylation, each sample was digested with trypsin (w[trypsin] : w[protein] = 1 : 20) at 37°C overnight. The tryptic peptides were labeled with the iTRAQ reagents; the AAD and control groups were labeled with iTRAQ 113 and 114, respectively. The peptides were pooled and desalted with Sep-Pak Vac C18 (Waters, Milford, MA, USA). The peptide mixture was diluted with Buffer A containing 10 mM KH2PO4 in 25% acetonitrile (ACN) at pH 2.6. The peptides were fractionated by a 20AD high-performance liquid chromatography (HPLC) system (Shimadzu) equipped with polysulfoethyl A column (2.1 mm × 100 mm, 5 μm, 200 A, The Nest Group, Southborough, MA, USA). The composition of Buffer B was 350 mM KCl, 10 mM KH2PO4, and 25% ACN at pH 2.6. Separation was performed using a linear binary gradient of 0–80% Buffer B in Buffer A at a flow rate of 200 μL/min for 1 hour. The fractions were combined into 20 groups.
3
Protein Digestion by FASP Method
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Protein digestion was performed using the FASP method [40 (link)]. A total of 300 µg proteins from each sample were placed on an ultrafiltration filter (30 kDa cutoff, Sartorius, Gottingen, Germany) that had 200 µL UA buffer (8 M urea, 150 mM Tris–HCl, pH 8.0). It was then centrifuged at 14,000g for 30 min and washed with 200 µL of UA buffer. About 100 µL of 50 mM iodoacetamide was added to the filter to block reduced cysteine residues. The samples were maintained at room temperature for 30 min in the dark, followed by centrifugation at a speed of 14,000g for 30 min. UA buffer (100 µL) was used to wash the filters twice. Approximately 100 µL of a dissolution buffer (Applied Biosystems, Foster City, CA, USA) was placed on the filter. This was centrifuged at 14,000g for 20 min, and then repeated twice. The protein suspensions were subjected to enzyme digestion with 40 µL of trypsin (Promega, Madison, WI, USA) buffer (4 µg trypsin in 40 µL of dissolution buffer) for 16–18 h at 37 °C. The final filter unit was transferred to a new tube that was spun at 14,000g for 30 min. The peptides were collected as a filtrate and the concentration of the peptides was measured at an optical density with a 280 nm wavelength (OD280).
4
Protein Digestion via FASP Method
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Protein digestion was conducted using the FASP procedure (Wisniewski et al., 2009 (link)). In brief, 300 mg of proteins were loaded onto an ultrafiltration filter (30 kDa cutoff; Sartorius, Goettingen, Germany) containing 200 µL of UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0), centrifuged at 14,000× g for 30 min, and then washed with 200 µL of UA buffer. Then, 100 µL of 50 mM iodoacetamide in UA buffer was subsequently added to the filter to block reduced cysteine residues. The samples were incubated for 30 min at room temperature in the dark and then centrifuged at 14,000× g for 30 min. The filters were washed three times with 100 µL of UA buffer and then centrifuged at 14,000× g for 30 min after each washing step. Next, 100 µL of dissolution buffer (Applied Biosystems, Foster City, CA, USA) was added to each filter and then centrifuged at 14,000× g for 30 min, which was repeated three times. The protein suspensions were then digested with 40 µL of trypsin (Promega, Madison, WI, USA) buffer (6 µg trypsin in 40 µL of dissolution buffer) at 37 °C for 18 h. Finally, the filter unit was transferred to a new tube, and 40 µL of dissolution buffer was added followed by centrifugation at 14,000× g for 30 min. The resulting peptides were collected as a filtrate, and the peptide concentration was analyzed at OD280.
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