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RNA Binding
Protein
Immunoprecipitation
Principle & Protocol

RNA is unstable and
needs to be bound to a
specific RNA-binding
protein.
Why How Definition
Introduction1
Goal
Aynamic association
between RNA and
RNA-binding proteins
accompanies the
entire life cycle.
RNA binding protein
Immunoprecipitation
(RIP)
based on RNA-binding
proteins to isolate or
discover functional
RNA molecules is a
common research
method for RNA
research.
RIP assay utilizes
antibodies against target
protein to precipitate the
corresponding RNA-
protein complexes.
The RNA bound to the
complex can be
validated by q-PCR or
sequencing analysis
after isolation and
purification.
Study the combination
of RNA and protein in
cells.
Understand the dynamic
process of post-
transcriptional regulation
of the network.
Help to discover the
regulatory targets of
miRNAs.

2 Workflow
RBP = RNA binding protein
+
RNP = Ribonucleoprotein complex
Analysis

3 Buffer Reagent
Polysome
lysis
buffer
NT2
buffer
100 mM KCl
5 mM MgCl2
10 mM HEPES (pH 7.0)
0.5% NP40
1 mM Dithiothrectol
100 units ml−1 RNase Out
400 μM VRC Protease inhibitor cocktail
50 mM Tris-HCl (pH 7.4)
150 mM NaCl
1 mM MgCl2
0.05% IGEPAL

Step 1
Preparation of mRNP lysate
4 Procedure of RIP
Step 2 Step 3
Antibody coating of
protein A/G beads
Immunoprecipitation
reaction and RNA
precipitation

1. Collect enough tissue culture cells to generate 2-5 mg of total protein per
RIP
2. Pellet by centrifugation for 10 min at 4 ℃
4.1 Preparation of mRNP lysate
3. Wash several times with 10 ml of ice cold PBS in a conical tube
The total amount of protein used per RIP must be optimized based upon the abundance of
the RBP and the planned method of RNA detection.Note
4. Resuspend cell pellet with polysome lysis buffer supplemented with RNase inhibitors and protease
inhibitors
5. Break cell clumps by pipetting up and down several times
6. Incubate mRNP lysate on ice for 5 min and store at -80℃
Note
Immediate freezing of the lysate is essential to complete the lysis process and prevent
adventitious binding.
Additional freeze-thaw cycles should be avoided to prevent protein and RNA degradation.
4 Procedure of RIP

4.2 Antibody coating of protein A/G beads
10. Wash antibody-coated beads with 1 ml of
ice-cold NT2 buffer to remove unbound
antibody and contaminants.
11. Resuspend beads in 850 μl of ice-cold
NT2 buffer.
8. In microcentrifuge tube, add 250-500 μl of
protein A-BSA slurry to yield pelleted beads after
a pulse centrifugation.
+
9. Add antibody to bead slurry and incubate
for 2-18 hours, tumbling end over end at 4
℃.
In parallel, a control antibody must be
used to assess background RNA levels.
Note
+
7. At 4 ℃, supplement pre-swell protein-
A/G Sepharose with 5% BSA to a final ratio
of 1:5 for at least 1 h before use.
BSA
4 Procedure of RIP

4.3 Immunoprecipitation reaction and RNA precipitation
12. Add cleared lysate to
antibody mixture prepared in
Step 11.
+
Immunoprecipitation reaction
14. Incubate for 4 h at 4 ℃
tumbling end over end or
incubate 2 h at room
temperature.
13. Flicking tube to mix, centrifuge
briefly to pellet beads. Remove
supernatant to obtain total cellular
mRNA.
++
Mix
15. Pellet beads and save
supernatant for later analysis
if desired.
+
4 Procedure of RIP

16. Wash beads with ice-cold NT2 buffer
by pulsing in an ultracentrifuge and
removing supernatant with a hand pipettor
or an aspirator.
+
17. Resuspend the beads in NT2 buffer
and incubate mixture for 30 min at 55 ℃,
flicking the tube occasionally using a
finger.
+
Resuspend
18. Add either Trizol reagent or phenol-
chloroform-isoamyl alcohol directly to
the beads to release RNP components
and isolate the RNA.
Trizol reagent
Phenol-chloroform-isoamyl alcohol
Release RNP
components
Isolate RNA
4.3 Immunoprecipitation reaction and RNA precipitation
4 Procedure of RIP

Microarray
qPCRRT-PCR
Sequencing
4.4 Analysis
4 Procedure of RIP

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