To visualise each sub-proteome, 20 μg of each sample was loaded onto a 1D SDS-PAGE. To determine the amount of intracellular contamination in the extracellular proteome, the activity of an intracellular enzyme marker glyceraldehyde phosphate dehydrogenase (GAPDH) was assayed on each sub-proteome as per the manufacturer's instructions (Sigma, St Louis). Five hundred microgram of each sample was resuspended in 250 uL 0.5 M triethylammonium bicarbonate (pH 8.5) before reduction and alkylation with 25 uL of 50 mM tris (2-carboxyethyl)phosphine (Thermo Scientific, Waltham) and 12.5 uL 200 mM methyl methanethiosulfonate (Sigma, St Louis), respectively. Samples were digested overnight at 37°C with trypsin (Sigma, St Louis) at a ratio of 1:10, subsequently desalted on a Strata-X 33 um polymeric reverse phase column (Phenomenex, Torrance, CA, USA) and dried in a vacuum centrifuge.
Strata x 33 um polymeric reverse phase column
Manufactured by Phenomenex
Sourced in United States
Strata-X 33 um polymeric reverse phase column is a high-performance chromatography column designed for the separation and analysis of a wide range of compounds. It features a 33 μm particle size and a polymeric reverse phase material, which provides high-resolution separations and excellent peak shape across a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Merck Group (4 mentions)
Methyl methanethiosulfonate, by Merck Group (4 mentions)
Tris 2 carboxyethyl phosphine, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Biosprint shaker, by Qiagen (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
4 protocols using strata x 33 um polymeric reverse phase column
1
Intracellular Contamination Detection in Extracellular Proteome
2
Proteomic Analysis of Fungal Biomass
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Dried mycelia and zoospores were ground to a fine powder using beads and Biosprint shaker (Qiagen). 300 μL of extraction buffer (25 mM Tris-HCl pH 7.5, 0.25% SDS, 50 mM Na 2 PO 4 , 1 mM Na 2 F, 50 μM Na 3 VO 4 and 1 mM PMSF in the presence of a protease inhibitor cocktail (Sigma)) was added to the total cell lysates and incubated for 30 min on ice with occasional vortexing. Samples were centrifuged at 15000g for 30 min at 4 • C and the supernatant was transferred to a new tube. The dried extracellular proteins were resuspended in 3 mL of water and proteins from the three sub-proteomes were precipitated with 6 volumes of acetone. Samples were initially digested with trypsin for 3 h at 37 • C to assist in solubilising the pellet, reduced and alkylated with 50 mM tris (2-carboxyethyl)phosphine (Thermo Scientific, Waltham) and 200 mM methyl methanethiosulfonate (Sigma, St Louis) respectively. Samples were digested again overnight at 37 • C with trypsin (Sigma, St Louis) at a ratio of 1:10, subsequently desalted on a Strata-X 33 um polymeric reverse phase column (Phenomenex, Torrance, CA, USA) and dried in a vacuum centrifuge.
3
Intracellular Protein Profiling by SDS-PAGE
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To visualise each sub-proteome, 20 μg of each sample was loaded onto a 1D SDS-PAGE. To determine the amount of intracellular contamination in the extracellular proteome, the activity of an intracellular enzyme marker glyceraldehyde phosphate dehydrogenase (GAPDH) was assayed on each sub-proteome as per the manufacturer's instructions (Sigma, St Louis). 500 ug of each sample was resuspended in 250 uL 0.5 M triethylammonium bicarbonate (pH 8.5) before reduction and alkylation with 25 uL of 50 mM tris(2-carboxyethyl)phosphine (Thermo Scienti c, Waltham) and 12.5 uL 200 mM methyl methanethiosulfonate (Sigma, St Louis) respectively. Samples were digested overnight at 37°C with trypsin (Sigma, St Louis) at a ratio of 1:10, subsequently desalted on a Strata-X 33 um polymeric reverse phase column (Phenomenex, Torrance, CA, USA) and dried in a vacuum centrifuge.
4
Mycelial Proteome Extraction and Digestion
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Dried mycelia and zoospores were ground to a fine powder using beads and Biosprint shaker (Qiagen). 300 µl of extraction buffer (25 mM Tris-HCl pH 7.5, 0.25% SDS, 50 mM Na2PO4, 1 mM Na2F, 50 µM Na3VO4 and 1 mM PMSF in the presence of a protease inhibitor cocktail (Sigma)) was added to the total cell lysates and incubated for 30 min on ice with occasional vortexing. Samples were centrifuged at 15000 g for 30 minutes at 4 °C and the supernatant was transferred to a new tube. The dried secretome was resuspended in 3 mL of water and proteins from the three sub-proteomes were precipitated with 6 volumes of acetone. Samples were initially digested with trypsin for 3 hours at 37 °C to assist in solubilising the pellet, reduced and alkylated with 50 mM tris (2-carboxyethyl)phosphine (Thermo Scientific, Waltham) and 200 mM methyl methanethiosulfonate (Sigma, St Louis) respectively. Samples were digested again overnight at 37°C with trypsin (Sigma, St Louis) at a ratio of 1:10, subsequently desalted on a Strata-X 33 um polymeric reverse phase column (Phenomenex, Torrance, CA, USA) and dried in a vacuum centrifuge.
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