Isobaric labeling of digested protein lysates was performed using the 10-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific). TMT reagents (0.8 mg) were dissolved in 41 μl of dry acetonitrile (ACN), and 7.5 μl was added to 15 μg of digested lysate in 100 mM TEAB. After 1 hour incubation at room temperature, the reaction was quenched by adding 3 μl of 5% hydroxylamine. Labeled peptides were combined and dried for the subsequent high pH reverse phase peptide fractionation (Thermo Fisher Scientific). Eight fractions were generated for each combined set.
High ph reverse phase peptide fractionation
Manufactured by Thermo Fisher Scientific
High pH reverse phase peptide fractionation is a laboratory technique used to separate and purify peptides based on their hydrophobicity. The process involves the use of a high pH mobile phase and a reversed-phase column to achieve effective separation of complex peptide mixtures.
Automatically generated - may contain errors
Lab products found in correlation
S trap micro column, by Protifi (2 mentions)
Peptide quantification assay, by Thermo Fisher Scientific (2 mentions)
Qubit protein quantification assay, by Thermo Fisher Scientific (2 mentions)
11 plex tandem mass tag reagents, by Thermo Fisher Scientific (2 mentions)
10 plex tandem mass tag reagents, by Thermo Fisher Scientific (1 mentions)
3 protocols using high ph reverse phase peptide fractionation
1
Isobaric Labeling of Digested Proteins
2
Subcellular Protein Fractionation and TMT Labeling
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5x106 frozen cell pellets from each condition (n=5) were used to perform subcellular protein fractionation. Minute Cytoplasmic and Nuclear Extraction kit (Invent Biotech, MN) was used to prepare nuclear and cytoplasmic fractions according to the manufacturer’s instruction. Protein concentration was measured by Qubit Protein Quantification Assay (Thermo Fisher, Rockford, IL). 35μg of protein lysate (nuclear or cytoplasmic) from each sample were trapped and digested by trypsin on a micro S-trap column (PROTIFI, NY) according to the manufacturer’s instruction. 0.6μg of the peptide mixture was used for peptide quantification assay (Thermo Fisher, Rockford, IL).
Isobaric labeling of peptides was performed using the 11-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific, Rockford, IL). TMT reagents (0.8mg) were dissolved in 41μl of dry acetonitrile (ACN), and 20.5μl was added to 25μg of digested peptide in 100mM TEAB. After 1 hour incubation at room temperature, the reaction was quenched by adding 4.2μl of 5% hydroxylamine. Labeled peptides were combined and dried for the subsequent high pH reverse phase peptide fractionation (Thermo Fisher, Rockford, IL). 8 fractions were generated for each combined set. Each TMT labeled fraction was reconstituted in a solution of 2 % acetonitrile (ACN), 2 % formic acid (FA) for MS analysis.
Isobaric labeling of peptides was performed using the 11-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific, Rockford, IL). TMT reagents (0.8mg) were dissolved in 41μl of dry acetonitrile (ACN), and 20.5μl was added to 25μg of digested peptide in 100mM TEAB. After 1 hour incubation at room temperature, the reaction was quenched by adding 4.2μl of 5% hydroxylamine. Labeled peptides were combined and dried for the subsequent high pH reverse phase peptide fractionation (Thermo Fisher, Rockford, IL). 8 fractions were generated for each combined set. Each TMT labeled fraction was reconstituted in a solution of 2 % acetonitrile (ACN), 2 % formic acid (FA) for MS analysis.
3
Subcellular Protein Fractionation and TMT Labeling
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