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.
Biosprint shaker
Manufactured by Qiagen
The Biosprint shaker is a laboratory equipment designed for gentle mixing and agitation of samples. It provides a consistent and controlled shaking motion to facilitate various laboratory processes that require gentle sample handling.
Automatically generated - may contain errors
Lab products found in correlation
Strata x 33 um polymeric reverse phase column, by Phenomenex (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Merck Group (2 mentions)
Tris 2 carboxyethyl phosphine, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Methyl methanethiosulfonate, by Merck Group (2 mentions)
2 protocols using biosprint shaker
1
Proteomic Analysis of Fungal Biomass
2
Mycelial Proteome Extraction and Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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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