Sulforhodamine bazide

A total of 405 ng of either PARP10_cat or
PARP15_cat and 1.4 μg of SPRK2 were
incubated with NAD⁺ (0, 1, 10, 50, 100
μM) in reaction buffer (50 mM Tris-HCl pH 8.0,
100 mM NaCl, 4 mM MgCl2, 0.5 mM TCEP·HCl) at 30 °C for 1 h.
The reaction was quenched by the addition of 10% SDS to a final
concentration of 2%. A 3.5× stock of AO-alkyne at pH 5 (525
mM acetate, 787.5 mM NaCl, 35 mM PDA) was added to the reaction mixture
(final [AO-alkyne] = 100
μM) and incubated at room temperature for 1 h. A
4.5× stock of CB (450 μM of TBTA, 4.5 mM
CuSO4, 450 μM rhodamine-azide (Sulforhodamine B
Azide, Click Chemistry Tools), 4.5 mM TCEP·HCl in 1× PBS
with 1% SDS) was added to the reaction mixture (final
[rhodamine-azide] = 100 μM)
and incubated at room temperature for 30 min. A 4× sample buffer
(with 5% BME) was added prior to fractionation by SDS-PAGE, and
subsequent fluorescence detection was performed using a ChemiDoc MP Imaging
System (Bio-Rad). Coomassie staining of the same gel was performed after
fluorophore visualization.

Cells were labeled with 1 as above. The medium was
removed and replaced with fresh medium for 5 min. Cells were washed with
1× PBS and fixed with 4% PFA in 1× PBS for 15 min at
room temperature. The cells were washed 3 × 5 min with 1×
PBS. Cells were permeabilized with 0.2% Triton X-100 in 1×
PBS for 5 min at room temperature followed by washing with 1× PBS
for 5 min. The cells were blocked in 3% BSA in 1× PBS at 4
°C for 2 h. The Click-iT Cell Reaction Buffer Kit (Life
Technologies) was used for in-cell click conjugation of rhodamine-azide (1
μM, Sulforhodamine B Azide, Click Chemistry
Tools). Cells were washed 2× with 3% BSA and
ddH₂O. Coverslips were mounted with ProLong Gold Antifade
Mountant with DAPI (Life Technologies). Images were taken on a Zeiss ApoTome
wide-field microscope with the following channel parameters: blue, Ex:
380/40, Em: 445/50; green, Ex: 470/40, Em: 525/50; red: Ex: 545/ 25, Em:
605/70 and objectives: 20 × 0.8 PlanApo, 60 × 1.4 PlanApo.
Images were processed in Zen 2012 (Zeiss) and levels were linearly adjusted
in Adobe Photoshop CS5.