( ly for Western analysis but...)Efficient Cellular Fractionation for RNA ,,, and Protein Isolation

ly for Western analysis, but also for functional or enzymatic assays. Thus, we were able to directly measure luciferase activity in total, nuclear, and cytoplasmic lysates prepared from HeLa cells transfected with firefly luciferase mRNA (Figure 3). As expected, no detectable luciferase activity was observed in the nuclear fraction, confirming the efficient cellular fractionation previously determined by Western blot analysis in Figure 1 and RT-PCR in Figure 2.

Figure 3. Luciferase Activity in HeLa Cells Lysates. 3x10⁵ HeLa cells were transfected in duplicate in 6 well plate with 1 g of luciferase mRNA. The relative luciferase activity in each protein fraction prepared with the PARIS Kit was measure 18 hours after transfection. Luciferase mRNA was prepared with Ambion's mMESSAGE mMACHINE and Poly(A) Tailing Kits.

2D Protein Analysis
To further examine the distribution of a large number of endogenous proteins, we performed two-dimensional (2D) gel electrophoresis with total, nuclear, and cytoplasmic protein lysates from HeLa cells (Figure 4). To remove genomic DNA that can interfere with the 2D analysis, the protein lysates were first treated with TURBO DNase (patent pending), a hyperactive DNase I mutant compatible with the Cell Disruption and Cell Fractionation Buffers. A direct comparison between each gel showed that 1) only few proteins were present in both nuclear and cytoplasmic fractions and 2) all of the nuclear or cytoplasmic proteins were also present in the total protein sample (compare Total and Nuclear/Cytoplasmic overlay in F
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