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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

2D GEL ASSAY FOR RNA-BINDING PROTEINS

2D Gel Assay for RNA-Binding Proteins
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
This protocol requires previous separation of RNA-protein complexes by native polyacrylamide gel electrophoresis. This protocol resumes with the completion of the native gel electrophoresis.
 
Procedure
1. Cut out the polyacrylamide fragment containing the RNA-protein complex of interest and place the fragment on a glass plate sitting on ice.

2. Crosslink the RNA-protein complexes by placing a hand-held Ultraviolet (UV) (CAUTION, see Hint #1) lamp approximately 4 mm above the gel fragment containing the RNA-protein complexes and irradiate gel slice for 17 min (see Hint #2).

3. Digest the uncrosslinked RNA by adding 100 to 150 μl of 2.5 mg/ml RNase A in Native Gel Buffer to a 1.5 ml microcentrifuge tube.

4. Immerse the gel slice completely in the RNase A solution.

5. Incubate the tube at 37°C for 30 minutes.

6. Remove the excess RNase A/Native Gel Buffer and continue the incubation at room temperature for 30 minutes.

7. Add 50 μl of 2X SDS-PAGE Sample Loading Buffer to the tube and soak the gel slice in this Loading Buffer for 1 to 2 hr at room temperature with mixing or shaking.

8. Boil the sample for 5 min.

9. Load a SDS-Polyacrylamide gel by placing the gel slice directly into the well and carry out SDS polyacrylamide electrophoresis (see Hint #3 and see Protocol for SDS-PAGE). Carry out further analyses or dry down gel and expose to X-ray film for detection (see Protocol for Drying Gels and see Protocol for Autoradiography).

Solutions
10 mg/ml RNase A
2X SDS-PAGE Sample Loading buffer   125 mM Tris, pH 6.8
2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
20% (v/v) Glycerol
0.001% (w/v) Bromophenol Blue
4% (w/v) SDS
Native Gel Buffer   50 mM Tris
50 mM Glycine
 
BioReagents and Chemicals
SDS
Glycine
RNase A
2-Mercaptoethanol
Glycerol
Bromophenol Blue
Tris
DTT
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The optimal time of irradiation needs to be empirically determined. A hand-held UV lamp can be positioned directly above gel slice by using a ring stand and clamps. Always use protective gear when exposed to UV light.

3. Prepare "thick" gels using wider gel spacers to cast the SDS-Polyacrylamide gel. That allows the gel slice to fit nicely into the well. A longer than normal "stacking" gel will yield better resolution.