Protocols

RNase Protection and Immunoprecipitation of Nuclear Extracts

Posted on Tuesday, October 21, 2003

Description
RNase Protection and Immunoprecipitation of Nuclear Extracts


Procedure
A. Prepare Beads (for 15 reactions)

1. Resuspend 60 mg of Protein A-Sepharose CL-4B or 375 ìl Protein G-Sepharose in 7.5 ml of NET2 (see Hint #2).

B. Antibody Binding to the Beads (for 1 reaction)

1. Add less than or equal to 0.3 to 20 ìl of antiserum to 500 ìl of Protein A-Sepharose CL-4b or Protein G-Sepharose in NET2.

2. Rotate for about 1 hour at room temperature or overnight at 4°C.

3. Wash 3 times with NET2 (pellet in clinical centrifuge, setting 1 or about 20 sec in microcentrifuge).

4. Resuspend in 200 ìl of NET2 (see Hint #3).

C. Hybridization Reaction

1. Prepare the hybridization reaction by combining the following ingredients:

4 ìl of 1 M KCl

5 ìl of 100 mM MgCl2

5 ìl of 100 mM DTT

4 ìl of 100 mM ATP

2 ìl of 0.2 M Creatine Phosphate

20 ìl of Nuclear extract [about 7 mg/ml] or S100

40 ìl of Buffer D

20 ìl of Transcript (about 500,000 cpm)

The total reaction volume is 100 ul

2. Incubate for 15 min at 20°C or 30°C or on ice and then place reactions on ice.

3. Add 40 ìl of RNase T1 or 1 mg/ml RNase A and incubate for 5 min on ice.

4. Transfer the entire 140 ìl reaction volume to 200 ìl of antiserum-bead mixture.

5. Rotate the bead mixture for 1 hr at 4°C.

6. Centrifuge the bead mixture for 20 sec in a microcentrifuge at full speed.

7. Transfer 2 ìl of the supernatant to 326 ìl of NET2 Mix.

8. Add an equal volume of Phenol/SEVAG and centrifuge briefly in a microcentrifuge to separate the phases. Recover the aqueous phase.

9. Ethanol precipitate with 750 ìl of Ethanol and centrifuge at full speed for 5 min.

10. Remove the supernatant and rinse the pellet with 70% Ethanol.

11. Allow the pellet to air dry.

D. Washing the Immunoprecipitates

1. Add 750 ìl of cold NET2 to the packed beads.

2. Centrifuge the beads for 20 sec in microcentrifuge at full speed.

3. Aspirate off the supernatant using a drawn out Pasteur pipette (see Hint #4).

4. Repeat Steps D1 to D3 five times.

5. Resuspend the beads in 165 ìl of NET2 Mix (see Hint #5).

6. Add an equal volume of Phenol-SEVAG, centrifuge briefly in a microcentrifuge and recover the aqueous phase.

7. Add 750 ìl of Ethanol, mix by inversion and centrifuge for 5 min at full speed in a microcentrifuge.

8. Remove the Supernatant and allow the pellet to air dry

9. Resuspend pellet in 3 ìl of RNA Loading Buffer

10. Heat at 90°C for 90 sec.

11. Place the sample on ice for 10 sec.

12. Pulse in microcentrifuge to collect all of the sample at the bottom of the tube.

13. Load on a 15% to 20% 7 M Urea gel (see protocol for Running Urea Gels; see Hint #6).


Recipes
1 M KCl

70% (v/v) Ethanol

Phenol/SEVAG 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)


RNA Loading Buffer 0.05% (w/v) Xylene Cyanol FF
0.05% (w/v) Bromophenol Blue
10 M Urea


0.2 M Creatine Phosphate

NET2 Mix 20 ìg carrier RNA
10 mM MgCl2
NET2
185 mM Sodium Acetate


1 mg/ml RNase A

NET2 50 mM Tris, pH 7.4
0.05% NP-40
150 mM NaCl


100 mM ATP

100 mM DTT

100 mM MgCl2


Supplies


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

2. Optional: wash beads several times with NET2 in clinical centrifuge. Resuspend in 7.5 ml NET2.

3. Beads can be stored at 4°C.

4. Be careful not to aspirate the beads.

5. Optional: Centrifuge sample 20 sec in microcentrifuge (to pellet residual beads) and transfer supernatant to a new tube.

6. Run an RNA ladder as a size marker along side. It is not necessary to dry the gel. Expose the film with an intensifying screen overnight to several days.


Submitted by: manny440