Alexa fluor 488 azide

Fluorescein-azide was purchased from Lumiprobe. AlexaFluor488-azide and AlexaFluor647-azide were purchased from Life Technologies. TAMRA-azide and Sulforhodamine101-azide were purchased from Click Chemistry Tools. Cy5.5 azide was purchased from AAT Bioquest.

ADP-ribosylation of SRPK2 by PARP10_cat was performed as
outlined above using 810 ng of PARP10_cat and 2.8
μg of SRPK2 with 6-a-NAD⁺ at
100 μM. The reaction was quenched by the addition
of 10% SDS to a final concentration of 2%. A 3.5×
stock of AO-TAMRA (Aminooxy-5(6)-TAMRA, Cayman Chemical) in pH 5 buffer (525
mM acetate, 787.5 mM NaCl, 35 mM PDA) or a 3.5× mock solution
(containing all components except substitution of DMSO for AO-TAMRA) was
added to the reaction mixture (final [AO-TAMRA] =
100 μM). The reaction was incubated at room
temperature for 1 h. A 4.5× stock of CB (450
μM of TBTA, 4.5 mM CuSO₄, 450
μM AF488-azide (Alexa Fluor 488 Azide, Life
Technologies), 4.5 mM TCEP·HCl in 1× PBS with 1%
SDS) or a 4.5× mock solution (containing all components except
substitution of DMSO for AF488-azide) was added to the reaction mixture
(final [AF488-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. Imaging was performed on a Typhoon 9400 (GE
Healthcare Life Sciences) using recommended laser settings and filter sets
for AF488 (Ex, 488 nm; PMT, 400 V; Em, 526 nm SP) and TAMRA (Ex, 532 nm;
PMT, 400 V; Em, 580 nm BP 30 nm). Coomassie staining of the same gel was
performed after fluorophore visualization. Each experiment was repeated at
least twice; shown are representative images.

ITAM-derived peptides used in the FP assay and time-resolved fluorescence resonance energy transfer (TR-FRET) assay were prepared by the Howard Hughes Medical Institute (HHMI) University of California (UC), Berkeley, Core Facility. Tetramethylrhodamine (TAMRA)-labeled ITAM 2pY peptide used in the FP assay consisted of the following sequence: CGNQLpYNELNLGRREEpYDVLD. 2pY peptide was incubated with twofold molar excess of TCEP to reduce the cysteine residue. This peptide was then incubated with a threefold excess of TAMRA C5 maleimide at room temperature overnight. The labeling reaction was quenched with 10-fold excess of DTT. The labeled peptide was purified by reverse phase high-performance liquid chromatography (HPLC) and its identify confirmed by mass spectrometry. The Alexa Fluor 488–labeled 2pY peptide used in the TR-FRET assay consists of the following sequence: XGNQLpYNELNLGRREEpYDVLD, where X is propargyl-glycine. Alexa Fluor 488-azide (Life Technologies, Grand Island, NY) was incubated in a threefold molar excess over 2pY peptide with the addition of ascorbic acid and copper sulfate at room temperature overnight. The labeled peptide was then purified by reverse phase HPLC and its identity was confirmed by mass spectrometry.