Denatured samples were applied to pre-wet Vivacon Hydrosart® stabilized cellulose filters (30KDa molecular weight cutoff; Sartorius) connected to LoBind tubes for reduction and alkylation steps. Samples were reduced with 20 mM dithiothreitol (DTT; Sigma) in 8 M Urea buffer for 30 min at 37 °C, followed by alkylation of free cysteines with 50 mM iodoacetamide (IAA; Sigma) in 8 M urea buffer for 20 min at room temperature (RT) in the dark. The flowthrough was discarded, and filters were washed three times with 2 M urea in 50 mM ammonium acetate, pH 5.5 (2 M urea buffer) before digestion for 3 h at 37°C using rLys-C and trypsin from the AccuMAPTM Low pH Protein Digestion Kit (Promega). Tubes were subsequently centrifuged to collect tryptic peptides in the flowthrough. 20% acetonitrile (Biosolve) in 50 mM ammonium acetate pH 5.5 was added to the filter to reduce non-specific binding followed by a further centrifugation step. Finally, 1% acetic acid in LC-MS grade water (Biosolve) was added to the flowthrough to inactivate enzymes prior to storage of the sample at −80 °C until solid-phase extraction and analysis by LC-MS/MS.
Accumap low ph protein digestion kit
Manufactured by Promega
Sourced in United States
The AccuMAP Low pH Protein Digestion Kit is a laboratory product designed for the digestion of proteins in a low pH environment. It provides the necessary reagents and protocols to facilitate the breakdown of proteins under acidic conditions.
Automatically generated - may contain errors
2 protocols using accumap low ph protein digestion kit
1
Optimized Protein Denaturation and Digestion
2
Low pH Protein Digestion and Alkylation
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The AccuMAPTM Low pH Protein Digestion Kit (Promega, Madison, WI, USA) and SMART DigestTM Trypsin Kit (Thermo Fisher Scientific) were used in Methods 4 and 5. The digestion procedure was performed according to the corresponding kit protocol.
In Method 5, after digestion, reductive alkylation was performed as follows: DTT was added to a final concentration of 10 mM, and the solution was incubated at 57°C for 30 min. MIA was added to a final concentration of 20 mM, and the mixture was incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 20% FA (1.0 μL) and 11 mM DTT. The enzymatic digest was desalted using an Oasis HLB μElution plate. The elution was dried and dissolved in 50 μL of FA solution.
In Method 5, after digestion, reductive alkylation was performed as follows: DTT was added to a final concentration of 10 mM, and the solution was incubated at 57°C for 30 min. MIA was added to a final concentration of 20 mM, and the mixture was incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 20% FA (1.0 μL) and 11 mM DTT. The enzymatic digest was desalted using an Oasis HLB μElution plate. The elution was dried and dissolved in 50 μL of FA solution.
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