3. Quantify protein concentration in the extracts using the Pierce
660 nm protein assay. In our hands, with HEK293 cells, protein concentrations average around 1.2 μg/μL.
4. Wash streptavidin-coupled magnetic beads twice with RIPA
buffer. Then incubate 150 μg of each of the cell extracts with
~15 μL (see Note 7) of washed beads under rotation overnight
at 4
C or for 1 h at room temperature. Make sure that beads
and extracts are mixed well during rotation; if necessary,
increase the volume by addition of RIPA buffer. Store the
remaining cell extract for Western blot analysis.
5. Collect magnetic beads using a magnetic rack and transfer the
supernatant into pre-labeled and chilled tubes. The supernatant should be kept for Western blot analysis (Fig. 3d).
6. Wash beads with 1 mL volume of the following ice-cold solutions: twice with RIPA buffer, once with 1 M KCl, once with
0.1 M Na 2 CO 3 , once with 2 M urea in 10 mM Tris–HCl pH 8
(freshly prepared), and again twice with RIPA buffer.
7. Elute by adding 60 μL 3Â protein sample buffer supplemented
with 2 mM biotin and 20 mM DTT, followed by 10-min
incubation at 97
C under vigorous shaking (see Note 7).
Quickly spin down and place the tubes on the magnetic rack.
Collect the eluates in fresh pre-labeled tubes.
8. Analyze cell extracts, bead supernatant, and eluate by Western
blotting using streptavidin-coupled HRP (Fig. 3a–d). Western
blot for the APEX2-fusion protein should also be performed
on the cell extracts, to confirm its expression and detect possible proteolytic cleavage events (see Figs. 2c and 3f) (see Note 8).
Stain the nitrocellulose membranes by Ponceau S as a loading
control (Fig. 3a).
3.4 Processing
of Cells from
Preparative Plates
1. Transfer tubes with cell pellets from 15 cm preparative plates
from À80
C storage to ice and resuspend pellets in 800 μL of
cold RIPA buffer supplemented with 1 mM PMSF, 1Â protease inhibitors cocktail, 10 mM sodium azide, 10 mM sodium
ascorbate, and 5 mM Trolox (sodium ascorbate and Trolox
should be fresh and can be weighed and directly dissolved in
the RIPA buffer).
2. Incubate the resuspended cells in RIPA buffer on ice for 2 min,
and then clear the extracts by centrifugation at 15,000 Â g for
10 min at 4
C. Carefully transfer the clarified extract without
disturbing the precipitated debris into new pre-chilled tubes.
3. Quantify protein concentration of the extracts using the Pierce
660 nm protein assay. In our hands, with HEK293 cells, protein concentrations average around 3 μg/μL.
Proximity-CLIP
291
660 nm protein assay. In our hands, with HEK293 cells, protein concentrations average around 1.2 μg/μL.
4. Wash streptavidin-coupled magnetic beads twice with RIPA
buffer. Then incubate 150 μg of each of the cell extracts with
~15 μL (see Note 7) of washed beads under rotation overnight
at 4
C or for 1 h at room temperature. Make sure that beads
and extracts are mixed well during rotation; if necessary,
increase the volume by addition of RIPA buffer. Store the
remaining cell extract for Western blot analysis.
5. Collect magnetic beads using a magnetic rack and transfer the
supernatant into pre-labeled and chilled tubes. The supernatant should be kept for Western blot analysis (Fig. 3d).
6. Wash beads with 1 mL volume of the following ice-cold solutions: twice with RIPA buffer, once with 1 M KCl, once with
0.1 M Na 2 CO 3 , once with 2 M urea in 10 mM Tris–HCl pH 8
(freshly prepared), and again twice with RIPA buffer.
7. Elute by adding 60 μL 3Â protein sample buffer supplemented
with 2 mM biotin and 20 mM DTT, followed by 10-min
incubation at 97
C under vigorous shaking (see Note 7).
Quickly spin down and place the tubes on the magnetic rack.
Collect the eluates in fresh pre-labeled tubes.
8. Analyze cell extracts, bead supernatant, and eluate by Western
blotting using streptavidin-coupled HRP (Fig. 3a–d). Western
blot for the APEX2-fusion protein should also be performed
on the cell extracts, to confirm its expression and detect possible proteolytic cleavage events (see Figs. 2c and 3f) (see Note 8).
Stain the nitrocellulose membranes by Ponceau S as a loading
control (Fig. 3a).
3.4 Processing
of Cells from
Preparative Plates
1. Transfer tubes with cell pellets from 15 cm preparative plates
from À80
C storage to ice and resuspend pellets in 800 μL of
cold RIPA buffer supplemented with 1 mM PMSF, 1Â protease inhibitors cocktail, 10 mM sodium azide, 10 mM sodium
ascorbate, and 5 mM Trolox (sodium ascorbate and Trolox
should be fresh and can be weighed and directly dissolved in
the RIPA buffer).
2. Incubate the resuspended cells in RIPA buffer on ice for 2 min,
and then clear the extracts by centrifugation at 15,000 Â g for
10 min at 4
C. Carefully transfer the clarified extract without
disturbing the precipitated debris into new pre-chilled tubes.
3. Quantify protein concentration of the extracts using the Pierce
660 nm protein assay. In our hands, with HEK293 cells, protein concentrations average around 3 μg/μL.
Proximity-CLIP
291