Yeast Sch9 Phosphorylation Assay: Difference between revisions
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==Materials== | ==Materials== | ||
* NTCB dissolve to 7.5 mM in water ( | * NTCB dissolve to 7.5 mM in water (1.68 mg/mL) | ||
* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi | * '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi | ||
* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. | * '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. | ||
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# Incubate overnight at room temperature. | # Incubate overnight at room temperature. | ||
# Add lysis buffer and blot using HA antibodies. | # Add lysis buffer and blot using HA antibodies. | ||
==From Park et al== | |||
Log phase cells were resuspended in 1 ml ice cold 0.2 M NaOH containing 0.2 % β- | |||
mercaptoethanol (v/v) and incubated on ice for 10 min. Then 50 μl of trichloroacetic acid | |||
was added and samples were incubated on ice for 10 min. 1M Tris base was added, and | |||
samples were boiled in 2X SDS sample buffer. SDS-PAGE and transfer to nitrocellulose | |||
was performed using standard protocols. Proteins were blotted from a 6% gel and a 4-20% gel | |||