To prepare for the protocol, users should (1) make all the
buffers and store them in sterilized containers and (2) thaw/make
up ATP, tRNA, and creatine phosphate. Please note that steps 1–3
are best done in 4
C cold rooms while the rest of the protocol can
be done at room temperature. At a minimum, the microcentrifuge
needs to be chilled to 4
C and kept at that temperature during
steps 1–3. In addition, depending on the downstream mass spectrometry protocol, users may choose to add a reduction and alkylating step before digesting with trypsin; these additions will replace
step 11 and are discussed in further detail in the Notes section
below.
Steps 1–3 are done at 4
C.
1. Spin beads at 1,702 Â g for 5 min in a microcentrifuge and
remove supernatant (see Note 17).
2. Wash beads 2Â with 2 M NaCl, 1 mL each time. Each spin is at
958 Â g for 5 min in a microcentrifuge.
3. Wash beads 2Â with GFB-100.
The rest of the protocol is done at room temperature.
4. Add 1.5 mL binding solution (which contains the protein
lysate of interest) to the bead/RNA mix and let rotate at
room temperature for 90 min (see Note 18).
5. Spin samples at 958 Â g for 5 min in a microcentrifuge.
6. Remove supernatant and add 1 mL GFB-200 + 6 mM MgCl 2.
7. Let rotate for 5 min, and spin samples at 958 Â g for 5 min in a
microcentrifuge.
8. Repeat steps 6–7 three additional times for a total of four
washes.
9. Wash with 20 mM Tris–HCl [pH ¼ 7.5]; spin for 5 min at
958 Â g in a microcentrifuge.
10. Wash with 100 mM NH 4 HCO 3 ; spin for 5 min at 958 Â g in a
microcentrifuge.
11. Resuspend in 100 μL 100 mM NH 4 HCO 3 (see Note 19).
12. Add 35 μL of 6 ng/μL trypsin.
13. Incubate at 37
C overnight.
3.3.3 Isolating Enriched
Proteins
After trypsin digestion, the proteins of interest exist as peptide
fragments in the supernatant. The last portion of the protocol
separates the supernatant from the beads and RNA fragments and
concentrates the peptides into a form that is usable for downstream
mass spectrometry.
1. Spin down (5 min at 958 Â g in a microcentrifuge) and transfer
supernatant to a new tube.
Using Bead-Tagged RNAs to Identify RBPs
249
buffers and store them in sterilized containers and (2) thaw/make
up ATP, tRNA, and creatine phosphate. Please note that steps 1–3
are best done in 4
C cold rooms while the rest of the protocol can
be done at room temperature. At a minimum, the microcentrifuge
needs to be chilled to 4
C and kept at that temperature during
steps 1–3. In addition, depending on the downstream mass spectrometry protocol, users may choose to add a reduction and alkylating step before digesting with trypsin; these additions will replace
step 11 and are discussed in further detail in the Notes section
below.
Steps 1–3 are done at 4
C.
1. Spin beads at 1,702 Â g for 5 min in a microcentrifuge and
remove supernatant (see Note 17).
2. Wash beads 2Â with 2 M NaCl, 1 mL each time. Each spin is at
958 Â g for 5 min in a microcentrifuge.
3. Wash beads 2Â with GFB-100.
The rest of the protocol is done at room temperature.
4. Add 1.5 mL binding solution (which contains the protein
lysate of interest) to the bead/RNA mix and let rotate at
room temperature for 90 min (see Note 18).
5. Spin samples at 958 Â g for 5 min in a microcentrifuge.
6. Remove supernatant and add 1 mL GFB-200 + 6 mM MgCl 2.
7. Let rotate for 5 min, and spin samples at 958 Â g for 5 min in a
microcentrifuge.
8. Repeat steps 6–7 three additional times for a total of four
washes.
9. Wash with 20 mM Tris–HCl [pH ¼ 7.5]; spin for 5 min at
958 Â g in a microcentrifuge.
10. Wash with 100 mM NH 4 HCO 3 ; spin for 5 min at 958 Â g in a
microcentrifuge.
11. Resuspend in 100 μL 100 mM NH 4 HCO 3 (see Note 19).
12. Add 35 μL of 6 ng/μL trypsin.
13. Incubate at 37
C overnight.
3.3.3 Isolating Enriched
Proteins
After trypsin digestion, the proteins of interest exist as peptide
fragments in the supernatant. The last portion of the protocol
separates the supernatant from the beads and RNA fragments and
concentrates the peptides into a form that is usable for downstream
mass spectrometry.
1. Spin down (5 min at 958 Â g in a microcentrifuge) and transfer
supernatant to a new tube.
Using Bead-Tagged RNAs to Identify RBPs
249