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The Laboratory of Donald Rio at the University of California, Berkeley
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A. Prepare Beads (for 15 reactions)
1. Resuspend 60 mg of Protein A-Sepharose CL-4B or 375 μl 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 μl of antiserum to 500 μl 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 μl of NET2 (see Hint #3).
C. Hybridization Reaction
1. Prepare the hybridization reaction by combining the following ingredients:
4 μl of 1 M KCl
5 μl of 100 mM MgCl2
5 μl of 100 mM DTT
4 μl of 100 mM ATP
2 μl of 0.2 M Creatine Phosphate
20 μl of Nuclear extract [about 7 mg/ml] or S100
40 μl of Buffer D
20 μl 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 μl of RNase T1 or 1 mg/ml RNase A and incubate for 5 min on ice.
4. Transfer the entire 140 μl reaction volume to 200 μl 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 μl of the supernatant to 326 μl 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 μl 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 μl 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 μl 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 μl 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 μl 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).
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| 0.2 M Creatine Phosphate |
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| NET2 Mix |
| 20 μg carrier RNA
10 mM MgCl2
NET2
185 mM Sodium Acetate
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| 1 mg/ml RNase A |
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| NET2 |
| 50 mM Tris, pH 7.4
0.05% NP-40
150 mM NaCl
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| 100 mM ATP |
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| 100 mM DTT |
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| 100 mM MgCl2 |
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| 1 M KCl |
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| 70% (v/v) Ethanol |
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| Phenol/SEVAG |
| 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
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| RNA Loading Buffer |
| 0.05% (w/v) Xylene Cyanol FF
0.05% (w/v) Bromophenol Blue
10 M Urea
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Potassium Chloride
Sodium Chloride
Sodium Acetate
RNase A
Ethanol
Urea
Protein G-Sepharose
NP-40
Protein A-Sepharose
Magnesium Chloride
Isoamyl Alcohol
Creatine Phosphate
Chloroform
Phenol
RNase T1
Tris
Xylene Cyanol FF
Bromophenol Blue
DTT
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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.
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1. Parker KA, Steitz JA. Determination of RNA-Protein And RNA-ribonucleoprotein interactions by nuclease probing. Methods Enzymol 1989;180:454-68
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