that biotinylation is dependent upon APEX2, BP, and hydrogen peroxide; and (3) to probe how well localization of biotinylated proteins correlates to APEX2 localization.
7. It is recommended to calibrate per each APEX2-fusion protein
expressing cell line the optimal ratio of bead volume to cell
extract total protein. While insufficient quantity of beads may
result in loss of biotinylated material that remains in solution,
use of too many beads can increase the experiment background. Only ~15% of bound material is eluted from streptavidin beads upon incubation with protein-loading dye (see
Subheading 2.3, step 18, and Subheading 3.3, step 7). Therefore, Western blot analysis of the unbound material after incubation of cell extracts with the beads may be the best indication
for full depletion of biotinylated proteins from the cell extracts
(Fig. 3c, d). Finally, when working with beads or resin, make
sure that either wide or cut tips are used to maintain their
integrity.
8. Seeding three 6 cm plates per cell line enables biochemical
control of the proximity biotinylation (Fig. 3). Western blot
analysis of cell extracts and eluates with HRP-coupled streptavidin controls for the dependence of the reaction in APEX2,
BP, and hydrogen peroxide. Similarly, analysis of the unbound
material and eluates will confirm that biotinylated proteins
were fully collected from the cell extracts and are concentrated
on the beads. Finally, Western blot analysis to confirm the
expression of the APEX2-fusion protein is vital to rule out
the expression of truncated versions of the protein.
9. The radiolabeled 19 nt and 35 nt RNA size markers (Table 1)
are essential as size markers on urea-PAGE gels, for RNA
footprint size selection, and for monitoring 3
0 and 5
0 adapter
ligations. We recommended loading non-ligated size markers
on all urea-PAGE gels, as well as introduction of the RNA size
markers into 3
0 ligation reaction mix, and their carryover
through gel extraction into the 5
0 ligation reaction. Radiolabel
10 pmol of each size marker individually at 37
C for 15 min in
10 μL reaction volumes of PNK buffer with DTT, 10 U T4
PNK, and 50 μCi γ32
P-ATP. Terminate the reaction by addition of 10 μL 2Â formamide gel loading solution and incubation at 90
C for 1 min. To purify the labeled markers from the
free γ32
P-ATP load and run them through a 15% urea-PAGE
with at least one empty lane between samples. Optional: To
reduce the amount of radioactive waste a small RNA column
purification kit (we use Zymo) can be used to clear the reaction
mix off the γ32 P-ATP prior to gel separation. Image the gels
by autoradiography, excise, extract, purify, and solubilize each
labeled marker in 10 μL of water. Finally, pool the markers
together; if the radioactive signal of the markers appeared
304
Daniel Benhalevy and Markus Hafner
7. It is recommended to calibrate per each APEX2-fusion protein
expressing cell line the optimal ratio of bead volume to cell
extract total protein. While insufficient quantity of beads may
result in loss of biotinylated material that remains in solution,
use of too many beads can increase the experiment background. Only ~15% of bound material is eluted from streptavidin beads upon incubation with protein-loading dye (see
Subheading 2.3, step 18, and Subheading 3.3, step 7). Therefore, Western blot analysis of the unbound material after incubation of cell extracts with the beads may be the best indication
for full depletion of biotinylated proteins from the cell extracts
(Fig. 3c, d). Finally, when working with beads or resin, make
sure that either wide or cut tips are used to maintain their
integrity.
8. Seeding three 6 cm plates per cell line enables biochemical
control of the proximity biotinylation (Fig. 3). Western blot
analysis of cell extracts and eluates with HRP-coupled streptavidin controls for the dependence of the reaction in APEX2,
BP, and hydrogen peroxide. Similarly, analysis of the unbound
material and eluates will confirm that biotinylated proteins
were fully collected from the cell extracts and are concentrated
on the beads. Finally, Western blot analysis to confirm the
expression of the APEX2-fusion protein is vital to rule out
the expression of truncated versions of the protein.
9. The radiolabeled 19 nt and 35 nt RNA size markers (Table 1)
are essential as size markers on urea-PAGE gels, for RNA
footprint size selection, and for monitoring 3
0 and 5
0 adapter
ligations. We recommended loading non-ligated size markers
on all urea-PAGE gels, as well as introduction of the RNA size
markers into 3
0 ligation reaction mix, and their carryover
through gel extraction into the 5
0 ligation reaction. Radiolabel
10 pmol of each size marker individually at 37
C for 15 min in
10 μL reaction volumes of PNK buffer with DTT, 10 U T4
PNK, and 50 μCi γ32
P-ATP. Terminate the reaction by addition of 10 μL 2Â formamide gel loading solution and incubation at 90
C for 1 min. To purify the labeled markers from the
free γ32
P-ATP load and run them through a 15% urea-PAGE
with at least one empty lane between samples. Optional: To
reduce the amount of radioactive waste a small RNA column
purification kit (we use Zymo) can be used to clear the reaction
mix off the γ32 P-ATP prior to gel separation. Image the gels
by autoradiography, excise, extract, purify, and solubilize each
labeled marker in 10 μL of water. Finally, pool the markers
together; if the radioactive signal of the markers appeared
304
Daniel Benhalevy and Markus Hafner