HEK293 cells in a similar growth stage 36 h prior to the
experiment. This sufficed in our hands for seeding one 15 cm
preparative plate, three 6 cm plates (+BP and hydrogen peroxide, and ÀBP and Àhydrogen peroxide controls) for Western
blot analyses (see also Note 8), and four cover glasses (ÀBP and
Àhydrogen peroxide controls and two +BP and hydrogen peroxide glasses) for immunofluorescence analyses. The additional
+BP and hydrogen peroxide glass is usually required for “no
primary antibody” or another technical control.
The compartment to which control APEX2 is targeted to
should be selected based on where signal from the compartment of interest is expected to diffuse to; often this would be
the cytoplasm. APEX2 fusion proteins should be detectable by
fluorescence microscopy, by immunostaining, or by fusion to a
fluorescent protein. Staining procedures should be optimized
beforehand (Fig. 2), and homogenic APEX2 expression as well
as proper localization in all expressing cells should be verified.
2. Although mass spectrometry is often outsourced, we recommend performing on-beads trypsinization and the preceding
steps in-house. Due to relatively low amounts of input material,
it is advisable to minimize the amount of added trypsin, to
strictly avoid contamination of samples with skin proteins, and
to use high-grade reagents. We used Pierce single-use/noweigh iodoacetamide and DTT (Thermo Scientific 90034
and 20291, respectively) and sequencing-grade modified trypsin (Promega V5111).
3. Urea-PAGE, autoradiography, and excision of gel sections to
obtain RNA:
Urea-polyacrylamide gels can be mixed and cast in-house.
We use premixed solutions (SequaGel UreaGel 29:1 Denaturing Gel System, by National Diagnostics) and cast 16 cm long
gels (length includes 2 cm for loading wells). Prior to loading
either pure RNA or a reaction mix on gel, samples should be
mixed in a 1:1 ratio with 2Â formamide gel loading solution
and incubated at 90
C for 1 min. As a rule of thumb, samples
should be loaded with at least one empty lane between them to
avoid cross-contamination and facilitate image analysis in case
autoradiography signals are dramatically different in their
intensity. Once gels are assembled on the apparatus, use a
syringe to wash wells with running buffer in order to remove
urea precipitations, and pre-run prior to loading the samples
for about 30 min to warm the gels. Repeat the wash of urea
precipitations immediately prior to loading your samples. Once
samples are loaded, we run our gels at 450 V for 45–70 min.
Once the run has ended, dismantle the gel while leaving it
attached to one of the glass plates. Mark three corners of the
gel at locations distant from the sample lane with ~0.1 μL of
302
Daniel Benhalevy and Markus Hafner
experiment. This sufficed in our hands for seeding one 15 cm
preparative plate, three 6 cm plates (+BP and hydrogen peroxide, and ÀBP and Àhydrogen peroxide controls) for Western
blot analyses (see also Note 8), and four cover glasses (ÀBP and
Àhydrogen peroxide controls and two +BP and hydrogen peroxide glasses) for immunofluorescence analyses. The additional
+BP and hydrogen peroxide glass is usually required for “no
primary antibody” or another technical control.
The compartment to which control APEX2 is targeted to
should be selected based on where signal from the compartment of interest is expected to diffuse to; often this would be
the cytoplasm. APEX2 fusion proteins should be detectable by
fluorescence microscopy, by immunostaining, or by fusion to a
fluorescent protein. Staining procedures should be optimized
beforehand (Fig. 2), and homogenic APEX2 expression as well
as proper localization in all expressing cells should be verified.
2. Although mass spectrometry is often outsourced, we recommend performing on-beads trypsinization and the preceding
steps in-house. Due to relatively low amounts of input material,
it is advisable to minimize the amount of added trypsin, to
strictly avoid contamination of samples with skin proteins, and
to use high-grade reagents. We used Pierce single-use/noweigh iodoacetamide and DTT (Thermo Scientific 90034
and 20291, respectively) and sequencing-grade modified trypsin (Promega V5111).
3. Urea-PAGE, autoradiography, and excision of gel sections to
obtain RNA:
Urea-polyacrylamide gels can be mixed and cast in-house.
We use premixed solutions (SequaGel UreaGel 29:1 Denaturing Gel System, by National Diagnostics) and cast 16 cm long
gels (length includes 2 cm for loading wells). Prior to loading
either pure RNA or a reaction mix on gel, samples should be
mixed in a 1:1 ratio with 2Â formamide gel loading solution
and incubated at 90
C for 1 min. As a rule of thumb, samples
should be loaded with at least one empty lane between them to
avoid cross-contamination and facilitate image analysis in case
autoradiography signals are dramatically different in their
intensity. Once gels are assembled on the apparatus, use a
syringe to wash wells with running buffer in order to remove
urea precipitations, and pre-run prior to loading the samples
for about 30 min to warm the gels. Repeat the wash of urea
precipitations immediately prior to loading your samples. Once
samples are loaded, we run our gels at 450 V for 45–70 min.
Once the run has ended, dismantle the gel while leaving it
attached to one of the glass plates. Mark three corners of the
gel at locations distant from the sample lane with ~0.1 μL of
302
Daniel Benhalevy and Markus Hafner