Electrophoretic Mobility Shift Assay
The Electrophoretic Mobility Shift Assay (EMSA), or gel mobility shift assay, is a popular and powerful technique for the detection of RNA–protein interactions. It relies on naked RNA having a certain mobility on non-denaturing gels. If the RNA is bound by protein, the mobility of the RNA is reduced. Thus, the binding of protein results in a characteristic upward shift of the RNA on a gel, as monitored using radio-labelled RNA. EMSAs can be performed using complex mixtures of proteins, such as cell extracts, and they can be used to investigate a wide range of RNA–protein interaction; single protein-binding events (simple interactions), small complexes formed from RNA, and the assembly of large multi-component RNA-protein complexes (for example, the spliceosome).
A. Reagents and Equipment
- Acrylamide (40% stock)
- Binding buffer for mobility shift assays
- Bisacrylamide (2% stock)
- Bovine serum albumin (BSA)
- Bromophenol blue
- Heparin (50 mg/mL [w/v] in H2O)
- Loading buffer for mobility shift assays
- Protein of interest (purified protein or cell extract)
- RNA of interest (uniformly labeled and purified)
- Splicing mix for mobility shift assays (4×)
- Total RNA or tRNA (10 mg/mL from yeast or Escherichia coli)
- Tris (pH 7.6)
- Tris/borate/EDTA (TBE) electrophoresis buffer (10×)
- Tris–glycine buffer (10×)
- Xylene cyanol
for Simple Interactions
- Label the RNA of interest. Tolabel the RNA uniformly with [α-32P] NTPs, use in vitro transcription. For anoligoribonucleotide or a 100–200-nucleotide purified RNA, end-label using [32P]cytidine 3’, 5’-bisphosphate or [γ-32P] ATP.
- Prepare the labeled RNA with purified protein under suitable binding conditions (e.g., binding buffer supplemented with 10 µg/mL BSA). A mixture of 20 µL Binding buffer(supplemented with 10 µg/mL BSA), 1 µL RNA (50,000 cpm) and 4 µL Purified protein(serial dilutions from excess to less than equimolar) on ice would be suitable.
- Incubate the mixture for 30–60min at room temperature, 30 ˚C, or 37 ˚C.
- Dilute the sample with 4 µL of loading buffer (without heparin) and load 10–20 µL of the mixture onto a native polyacrylamide gel. For a single protein (50,000 MW) and a single RNA (100 nucleotides long), start with a 0.8 mm-thick 6%acrylamide:bisacrylamide (30:1) gel made with Tris–glycine buffer or TBE. Pre-run the gel for 30 min.
- Perform electrophoresis at 300V for 3 h (or 13 V/cm), making sure that it does not heat up. If the interactions are suspected to be unstable, run the gel in a cold room.
- Use a sheet of Whatman 3MM paper to pick up the gel, and dry it at 80˚C.
- Expose the gel to a phosphorimager or X-ray film.
for Longer RNA Forming a Small Complex
- Prepare a labeled RNA target by in vitro transcription. Gel-purify the RNA to make sure that the substrate will be a single band on a gel.
- Incubate the labeled RNA with purified protein under suitable binding conditions. A mixture of 20 µL Binding buffer (supplemented with 10 µg/mL BSA), 1 µL RNA (50,000 cpm) and 1-4 µL Purified protein/cell extract on ice would be suitable.
- Incubate for 30–60 min at the preferred temperature (4˚C–37˚C).
- Following incubation, dilute the reaction to 100 µL with buffer containing 500 mM NaCl, 10 mM Tris (pH 7.6), 5% glycerol, and 200 µg/mL heparin (in cases where cellular extract is used).
- Incubate for 10 min at room temperature.
- Remove 1 µL of the reaction and add it to 30 µL of loading buffer. Make sure that the glycerol concentration is at least 5% so that the sample does not float away.
- Load this sample onto a 1.5 mm-thick, 20 cm-long 0.5× TBE, 6% acrylamide:bisacrylamide (60:1) gel. Perform electrophoresis at 25 mA until the xylene cyanol is about two-thirds down the gel.
- Disassemble the gel, pick it up with Whatman 3MM paper, and dry it at 80˚C.
- Expose the gel to a phosphorimager or X-ray film.
for Large Multi-Component RNA-Protein Complexes
- Prepare a native gel containing 3.75%–4.2% polyacrylamide and acrylamide:bisacrylamide (80:1).
- Pour the solution into 17 × 14.7 × 0.15 cm plates, insert a comb with 0.75 cm-wide wells, and allow the gel to polymerize. (This usually takes at least 30 min.)
- Mix the following components in a 1.5 mL microcentrifuge tube by pipetting up and down: 5 µL nuclear extract (15–20 mg/mL) or extract dialysis buffer, 1 µL RNA (25,000–50,000 cpm), 5 µL splicing mix (4×), 9 µL H2O
- Incubate for 30–60 min at 20˚C–30˚C.
- Stop the reaction by placing the tubes on ice. Add heparin (titrate a range between 5 and 500 µg/mL) to reduce nonspecific binding.
- Add 3 µL of 1% xylene cyanol and bromophenol blue to the reactions. Mix by pipetting up and down. Alternatively, the dye can be added to a free lane on the gel to monitor electrophoresis and prevent possible interference with protein binding.
- Pre-run the gel for 20 min at no more than 6 W.
- Load 5 µL of the binding reaction mixture from Step 5 onto the gel.
- Run the gel at no more than 6 W at 120–210 V maximum at room temperature until the dye is 1–2 cm from the bottom (for a 70-nucleotide RNA).
- Transfer the gel onto Whatman 3MM filter paper and dry it for 30–60 min at 80˚C on a vacuum gel dryer.
- Expose the gel to X-ray film with an intensifying screen at –80˚C.