Ultraviolet (UV) Photochemical Crosslinking to Detect RNA Bi

Sunday, October 19, 2003

Description
Labeled nucleotides from the RNA are covalently transferred to bound proteins following illumination by UV light. Excess RNA is then digested with Ribonuclease A and the labeled proteins are visualized by autoradiography following SDS polyacrylamide electrophoresis.

Procedure
1. This protocol requires RNA already labeled with radioactive nucleotide. (CAUTION! see Hint 31; also see protocol for Labeling RNA with [32P]).

2. Incubate the labeled RNA with the protein extract under the appropriate conditions for the desired time in a 1.5 ml microcentrifuge tube (see Hint #2).

3. Cover a centrifuge tube rack with a sheet of parafilm, depressing the film into each well slightly to leave a concave impression in the parafilm. This space will hold samples for irradiation with ultraviolet (UV) light. CAUTION! (see Hint #1)

4. Place the rack covered with the parafilm into an ice bucket filled with ice.

5. Pipette the samples onto the concave spaces on the parafilm. The samples will remain as small beads of liquid or "drops".

6. Irradiate the samples with UV light by placing a hand-held UV lamp (set at a wavelength of 254 nm) approximately 5 cm from the samples and irradiate for 12 minutes (see Hint #3). Make sure that the samples are directly facing the UV source and are not blocked from irradiation.

7. After irradiation, transfer the samples to new microcentrifuge tubes and add RNase A to a final concentration of 1 mg/ml.

8. Incubate samples at 30C for 30 minutes (see Hint #4).

9. After incubation, add 15 l of 2X SDS-PAGE Sample Loading Buffer and boil the samples for 5 minutes.

10. Load samples onto a 10% SDS-Polyacrylamide gel and electrophorese as usual (see Protocol on SDS-Polyacrylamide Electrophoresis).

11. Dry gel and expose the dried gel onto X-ray film. (see Protocol on Autoradiography).

Recipes
SDS-PAGE Sample Loading Buffer (2X) 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

Supplies

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

2. Optimal conditions for promoting the binding of proteins to RNA are empirically determined. Typical starting conditions are the assembly of RNA-protein complexes at room temperature for 15 minutes to 1 hour. Do not include polyvinyl alcohol (PVA) in the reaction, as PVA will interfere with SDS-PAGE analysis.

3. A "Stratalinker" can also be used to cross link the RNA and protein. The optimal time and distance from the UV light source should be empirically determined with appropriate titrations and variations.

4. Optimal concentrations of RNase A and/or mixtures of different RNases may need to be varied in order to achieve adequate digestion of non-crosslinked RNA.

(责任编辑：泉水)

搜索

热门标签:

Ultraviolet (UV) Photochemical Crosslinking to Detect RNA Bi