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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

NATIVE GEL ELECTROPHORESIS OF RNA-PROTEIN COMPLEXES

Native Gel Electrophoresis of RNA-Protein Complexes
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
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 μl 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 μl of Nuclear Extract (15 to 20 mg/ml)

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

0.625 μl of 0.2 M Creatine Phosphate

0.5 μl of 1 M Hepes-KOH, pH 7.6

0.5 μl of 150 mM MgCl2

0.75 μl of 100 mM ATP

5 μl of 50 mg/ml Heparin

11.7 μl of ddH2O

4. Mix the reaction gently a few times by trituration and incubate at 20°C 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 μl of each reaction, and load one lane with 3 μl 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 -80°C) (see Protocol on Autoradiography).

Solutions
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
0.2 M Creatine Phosphate
1 M Glycine
1 M Tris-HCl, pH 8.8
10% APS   10% (w/v) Ammonium Persulfate
 
BioReagents and Chemicals
Xylene Cyanol FF
APS
Creatine Phosphate
TEMED
Magnesium Chloride
Ammonium Persulfate
Tris
Bis-acrylamide
Acrylamide
HEPES
Glycine
Oligonucleotide
Bromophenol Blue
Heparin
ATP
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.