Yeast Sch9 Phosphorylation Assay: Difference between revisions
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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 | |||