Untreated or RNase-treated Krebs extracts were incubated with Luc or N1mΨ–Luc mRNAs (4 μg/ml) at 30°C. At the indicated times, 12.5 μl aliquots of the reaction mixtures were withdrawn and the translation was stopped by the addition of SDS-proteinase K solution (20 (link)). Following incubation for 15 min at room temperature, total RNA was extracted with phenol–chloroform and precipitated with ethanol. RNA was separated on formaldehyde-1% agarose gels and transferred onto nylon membranes (Hybond-N, GE Healthcare). To confirm equal RNA loading, the blots were stained with Blot Stain Blue (Sigma) and the intensities of bands of 18S ribosomal RNA (rRNA) were measured using NIH Image J. software. RNA was then hybridized with ∼300 bp-long fragment of randomly primed 32P-labeled luc cDNA using ExpressHyb hybridization solution (Clontech Laboratories, Inc), as described by the manufacturer. The blots were exposed to X-ray films. Bands of Luc mRNA were quantified using a Typhoon PhosphorImager (GE Healthcare).
Blot stain blue
Manufactured by Merck Group
Blot Stain Blue is a laboratory reagent used for the detection and visualization of proteins in Western blot analysis. It is a dye-based solution that binds to proteins on a membrane, allowing them to be visualized and quantified.
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2 protocols using blot stain blue
1
Krebs Extract RNA Translation Assay
2
Quantifying Luc mRNA Stability
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uRRLs (100 μl) were incubated with unmodified or Ψ-incorporated Luc mRNAs (4 μg/ml) at 30°C. At the indicated times, 15-μl aliquots of the reaction mixtures were withdrawn and supplemented with 200 μl of SDS/proteinase K solution (41 (link)). After incubation for 15 min at room temperature, total RNA was extracted with phenol-chloroform and precipitated with ethanol. RNA was resolved by formaldehyde–1% agarose gel electrophoresis and transferred onto nylon membranes (Hybond-N, GE Healthcare). To confirm that equal amounts of total RNA were loaded in each lane, the blots were stained with Blot Stain Blue (Sigma) and the intensities of bands of 28S ribosomal RNA (rRNA) were measured using NIH Image J software. RNA was then hybridized with a 44 nt-long 32P-labeled DNA oligo complementary to the Luc mRNA coding region using ExpressHyb hybridization solution (Clontech Laboratories, Inc), as described by the manufacturer. The blots were exposed to X-ray films. Bands of Luc mRNA were quantified using a Typhoon PhosphorImager (GE Healthcare).
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