Efficient Cellular Fractionation for RNA and Protein Isolation
|Ambion's Protein and RNA Isolation System, PARIS™, uses a rapid and simple procedure to isolate both total RNA and native protein from the same experimental sample. Cultured cells and tissues that are either fresh, frozen, or treated with RNA stabilization reagents such as RNAlater® and RNAlater®-ICE, are all compatible with the procedure. This kit provides a unique tool for researchers who need both RNA and protein for various downstream analysis, reducing time, cost, and variability between independent experimental samples. The PARIS Kit also permits separation of nuclear and cytoplasmic fractions from fresh cultured cells prior to RNA and/or protein isolation. Here, we tested the procedure with various mammalian cell types and show that both RNA and native protein are efficiently recovered from nuclear and cytoplasmic fractions with virtually no cross contamination between the two cellular compartments.|
Fast and Simple Cellular Fractionation
The PARIS Kit also contains a solution optimized for rapid nuclear/cytoplasmic fractionation of fresh cultured cells. The fractionation method is based on differential lysis of plasma and nuclear membranes by nonionic detergents. After selective lysis of the plasma membrane in Cell Fractionation Buffer, intact nuclei are collected by a quick centrifugation step and resuspended in Cell Disruption Buffer. The supernatant contains all of the cytoplasmic components. Following the same procedure described above, both protein and high quality RNA can then be isolated from each cellular fraction. The entire procedure for the simultaneous isolation of RNA and native protein from total or nuclear and cytoplasmic fractions can be completed in as little as 30 minutes.
Efficient Cellular Fractionation with Various Cell Types
Figure 1. Comparison of Total, Cytoplasmic, and Nuclear RNA and Protein From Different Cell Lines. RNA and protein were isolated from 1 x 106 cells using the PARIS Kit. To allow direct comparison between total (T), cytoplasmic (C), and nuclear (N) fractions, the same proportion (~5%) of each prep was analyzed by denaturing agarose gel electrophoresis (NorthernMax®-Gly Kit) or Western blot with anti-GAPDH antibody (Ambion). Nuclear vs cytoplasmic fractionation was performed in triplicate for 293 cells. M: Millennium Markers™.
Efficient cellular fractionation at the protein level was confirmed by Western blot analysis with an antibody specific for the cytoplasmic GAPDH protein (Figure 1, bottom panels). Equivalent amounts of GAPDH protein were present in the total and cytoplasmic fractions with no detectable cross contamination in the nuclear fraction for the four cell types tested.
Isolation of Intronless Cytoplasmic mRNA
Figure 2. Analysis of Fibronectin pre-mRNA Splicing. The mutually exclusive alternative splicing of fibronectin exon EIIIb was analyzed by RT-PCR (35 cycles) using total (T), cytoplasmic (C), and nuclear (N) RNA isolated from 293 cells with the PARIS Kit. Using an intron-specific primer, the unspliced form was detected only in the total and nuclear fractions. No PCR products were detected in the minus RT and minus RNA controls (data not shown).
Isolation of Native Protein
Figure 3. Luciferase Activity in HeLa Cells Lysates. 3x105 HeLa cells were transfected in duplicate in 6 well plate with 1 µg 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
Figure 4. 2D Analysis of Proteins Isolated Using the PARIS Kit. Total, nuclear, and cytoplasmic proteins (~25 µg) from HeLa cells were resolved using a pH 4-7 IPG gel followed by 8-16% SDS-PAGE. Gels were stained with SYPRO® Ruby (Molecular Probes). Panels on the right show a magnification of a portion of each gel and an overlay of the nuclear and cytoplasmic gel pictures.