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Download Current Protocols in Chemical Biology 2011 (Volume 3) by Adam P. Arkin PDF

By Adam P. Arkin

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This enables researchers to analyze the myristoylation or palmitoylation status of specific proteins of interest when combined with standard immunoprecipitation protocols on cells that have been metabolically labeled with Alk-C14, Alk-C16, or Alk-C18. In Basic Protocol 2, a cellular view of the distribution of myristoylated and palmitoylated proteins will be obtained (Fig. 3). The labeling method described here is very robust and the experimental results are typically consistent and reproducible across multiple runs.

Reagent quality and concentration Fatty acid probes were not used at the appropriate concentrations to obtain the best signal-to-noise ratio. Also, the click chemistry reaction works best only when the reagents are freshly prepared just prior to the experiment. Signal optimization Sometimes, staining is not homogeneous throughout the membrane blot. This may be due to uneven transfer of proteins from the gel. In this case, ensuring that the appropriate transfer procedure is used is critical. Also, if the intensity of bands on the membrane is high, then reducing exposure times or using a more diluted stock of streptavidin-HRP can resolve this issue.

Although there is no reported systematic analysis of the affinity of PUM-HD-RNA complex, see Cheong and Hall (2006) for additional information on the design of PUM-HD mutants. The applicability of PUM-HD to technology was first demonstrated for imaging mitochondrial mRNA, specifically NADH dehydrogenase 6 (ND6) mRNA. The HsPUM1-HD (hereafter named PUM1) was designed to match the RNA sequence of ND6, which was connected with EGFP fragments (Fig. 1B). Upon interacting with RNA, PUM1 brings the fragments of EGFP into an orientation and proximity in which they are sufficiently close for the fragments to associate and fluoresce.

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