RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction
Published: June 29, 2023
Abstract
Source: Kresoja-Rakic, J., et al. Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells. J. Vis. Exp. (2018)
This video demonstrates an in vitro RNA pull-down assay to identify RNA-binding proteins (RBPs), which interact with the adenylate-uridylate-rich element (ARE) sequences in mRNA. The target RBPs from a cell lysate are mixed with an RNA probe to form RNA-protein complexes. The complexes are isolated via affinity purification utilizing the affinity of the desthiobiotin label of the RNA probe to a streptavidin-labeled magnetic bead.
Protocol
1. Preparation of Cytosolic and Nuclear Protein Fraction Plate 4 x 106 ACC-MESO-4 cells in a T150 cell culture flask grown in RPMI – 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin (100x), and 2 mM L-Glutamine (200 mM). When cells reach a confluence of 80-90% (~5-5.5 x 106 cells), proceed with protein extraction of nuclear and cytosolic fractions. NOTE: ACC-MESO-4 cell line was obtained from the RIKEN BioResource Centre. <l…
Representative Results
Figure 1: Western blot analysis of 5 µg of cytosolic and nuclear protein fraction. PARP protein is present in the nuclear fraction (N) but absent from the cytosolic (C) fraction. α-tubulin is present in the cytosolic fraction (C) but absent in the nuclear fraction (N). Table 1: Sequences of RNA probes <table border="1" fo:keep-together.within-page="1" fo:keep…
Divulgations
The authors have nothing to disclose.
Materials
| NE-PER Nuclear and cytoplasmic extraction kit | Thermo Fisher Scientific | 78833 | |
| Pierce Protease Inhibitor Tablets, EDTA-free | Thermo Fisher Scientific | A32965 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
| Pierce Magnetic RNA-protein Pull-Down Kit | Thermo Fisher Scientific | 20164 | Includes magnetic beads and therefore the kit need to be stored at 4 °C |
| Pierce RNA 3’ End Desthiobiotinylation Kit | Thermo Fisher Scientific | 20163 | The kit is a part of the "Pierce Magnetic RNA-protein Pull-Down Kit", and needs to be stored at -20 °C unlike the pull-down kit (4 °C). |
| DynaMag-2, magnetic stand | Thermo Fisher Scientific | 12321D | |
| RPMI – 1640 medium | Sigma-Aldrich | R8758 | |
| FBS – Filtrated Bovine Serum | Pan Biotech | P40-37500 | |
| Penicillin – streptomycin (100x) | Sigma-Aldrich | P4333 | |
| L-Glutamine solution (200 mM) | Sigma-Aldrich | G7513 | |
| 0.25% Trypsin-EDTA (1x) | Gibco by Life technologies | 25200-056 | |
| RNaseZap RNase Decontamination Solution | Thermo Fisher Scientific | AM9780 | |
| RNA oligo synthesis | Mycrosynth AG, Switzerland | – | The synthesis scale – 0.04 µmol – HPLC-purified |
| Bovine serum albumin | Sigma-Aldrich | A7030 | |
| Hydrochloric Acid (HCl) 6 M | PanReac AppliChem | 182883.1211 | |
| Ultra Pure 1 M Tris, pH 7.5 | Thermo Fisher Scientific | 15567027 | |
| Glycerol | Thermo Fisher Scientific | 17904 | 50% sterile aliquots to be stored at -20°C |
| Pierce Streptavidin magnetic beads | Thermo Fisher Scientific | 88817 | Please note that these magnetic beads have an average diameter of 1 µm (range from 0.5 – 1.5 µm). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 µm, are also used in RNA-pulldown but be aware that this may affect purification process. |
| Aluminiumsulfate-(14-18)-hydrate | Sigma-Aldrich | 368458 | |
| Ortho-phosphoric acid | Applichem | A0637 |
Tags
Citer Cet Article
RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction. J. Vis. Exp. (Pending Publication), e21439, doi: (2023).