Native Gel Electrophoresis of RNA-Protein Complexes

Sunday, October 19, 2003

Description
This protocol is optimized to resolve complexes formed on short (50 to 70 nucleotide) RNA oligonucleotides. Incubation conditions, percentage of Acrylamide, and length of separation in the gel should be empirically determined for other complexes of interest.

Procedure
1. Prepare a 4.2% Polyacrylamide gel by combining the following:

7 ml of 30% Acrylamide:Bis-Acrylamide (60:1)

2.5 ml of 1 M Tris-HCl, pH 8.8

2.5 ml of 1 M Glycine

37.5 ml ddH2O.

2. Add 0.25 ml of 10% APS, mix, then add 25 l of TEMED, mix, and pour into 17 cm x 14.7 cm x 0.15 cm glass plates (see Hint #2). Insert a comb and allow the Acrylamide to polymerize for at least 30 min (see Hint #3).

3. Incubate the RNA in the previously prepared nuclear extract (see Protocols on Preparation of Drosophila Tissue Culture Cell Nuclear Extract and in vitro Transcription of RNA). In a 1.5 ml microcentrifuge tube on ice, add the following in order:

5 l of Nuclear Extract (15 to 20 mg/ml)

1 l of labeled RNA (25,000 to 50,000 cpm) (CAUTION! see Hint #1)

0.625 l of 0.2 M Creatine Phosphate

0.5 l of 1 M Hepes-KOH, pH 7.6

0.5 l of 150 mM MgCl2

0.75 l of 100 mM ATP

5 l of 50 mg/ml Heparin

11.7 l of ddH2O

4. Mix the reaction gently a few times by trituration and incubate at 20C for 30 to 60 min (see Hint #4).

5. Stop the reactions by placing tubes on ice.

6. Set up the Polyacrylamide gel (see Nucleic Acid Denaturing Gel Electrophoresis), and pre-run the gel for 20 min at 3 to 5 watts constant power.

7. Load the gel with 5 l of each reaction, and load one lane with 3 l of Dye Solution.

8. Run the gel at 120 to 210 Volts at room temperature until the Bromphenol Blue dye has migrated to 1 to 2 centimeters from the bottom of the gel (see Hint #5).

9. After electrophoresis, disassemble gel apparatus and wrap the gel in saran wrap. Expose gel to X-ray film to visualize complexes (1 to 4 hours with intensifying screen at -80C) (see Protocol on Autoradiography).

Recipes
0.2 M Creatine Phosphate

1 M Glycine

1 M Tris-HCl, pH 8.8

10% APS 10% (w/v) Ammonium Persulfate

30% Acrylamide:Bis-Acrylamide (60:1) (CAUTION! see Hint #1)

Dye Solution 1% (v/v) Xylene Cyanol FF
1% (w/v) Bromphenol Blue
in ddH2O.

50 mg/ml Heparin

100 mM ATP

150 mM MgCl2

1 M Hepes-KOH, pH 7.6

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. This protocol is designed for the use of a specific gel electrophoresis apparatus. Please adjust volume of Polyacrylamide gel accordingly for your own gel electrophoresis apparatus, and see the manufacturer's manual for specific instructions.

3. A comb with 0.75 cm wide wells can be used here.

4. Heparin is included to resolve complexes into discrete bands. Without heparin, some complexes can appear as a radioactive smear. Heparin also is known to strip some proteins from the complexes. Total RNA or tRNA can also be added to clean up the signal. To optimize conditions, titrate heparin to a fial concentration ranging from 1 to 25 mg/ml, and/or titrate total yeast RNA from 0.1 to 5 g per reaction.

5. Be sure not to overload the wells because of the poor stacking capabilities of the gel.

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Native Gel Electrophoresis of RNA-Protein Complexes