In this issue:

(1) γ-phosphate labeled ATP: Kinase substrate identification by non-radioactive in vitro Phosphorylation
(2) ARC-Fluo-/ARC-Lum Kinase Assays: High-throughput Screening of basophilic kinase inhibitors
(3) ATP-Agaroses: Affinity purification of recombinant kinases

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Kinase substrates are routinely identified by in vitro phosphorylation with radioactive ("hot") ATP whose modified gamma-phosphate (^{32 or 33}γP) is transferred by a kinase to a serine, threonine or tyrosine hydroxyl group of its peptide/protein substrate.

Lee et al.^[1] reported a non-radioactive version of in vitro phosphorylation were azide- and alkyne-γP-modified ATP analogs have been successfully used instead of 32γP-labeled ATP to phosphorylate p27^kip1 with protein kinase cdk2 (Fig. 1). The phosphorylated substrate can subsequently be labeled with Biotin or fluorescent dyes via Cu(I)- catalyzed or Cu(I)-free Click-Chemistry (Tab. 1).

Figure 1: GST-tagged recombinant p27kip1 was incubated with kinase Cdk2 in the presence of one of the following Azide/Alkyne γP-labeled ATP analogs: γ-[(Propargyl)-imido]-ATP (Cat. No. CLK-T11-1), γ-[(6-Azidohexyl)-imido]-ATP (Cat. No. CLK-T12-1) or γ-[2-Azidoethyl]-ATP (Cat. No. NU-1701). Phosphorylation was detected by immunoblotting with a phospho-p27^kip1 specific antibody^[1].

Table 1: Alkyne and Azide groups are detected via Cu(I)-catalyzed or Cu(I)-free Click Chemistry, respectively.

ATP analog	Cat. No.	Click Chemistry type	Labeling with
γ-[(Propargyl)-imido]-ATP	CLK-T11-1	Cu(I)-catalyzed	Azides of fluorescent dyes Azides of Biotin
γ-[(6-Azidohexyl)-imido]-ATP	CLK-T12-1	Cu(I)-free	DBCO-containing fluorescent dyes DBCO-containing Biotin
γ-[2-Azidoethyl]-ATP	NU-1701

ARC-based Protein Kinase Assays are designed to identify both ATP- and substrate competitive inhibitors of basophilic protein kinases from the AGC group (e.g. PKA, PKC) by competitive displacement of an innovative fluorescent probe (ARC-Fluo or ARC-Lum) from its complex with a protein kinase^{[2,3,4] (Fig. 2).}

ARC-based Protein Kinase Assays are amenable to automation (384-well microtiter plate format) and ideally suited for HTS experiments since they are

• reliable: Z-factor > 0.8 as determined with a PheraStar (BMG) platereader
• simple: 1 step mix & set up (homogenous assay)
• quick: 15 minutes incubation time
• cost-sensitive: no lanthanides, no antibodies, no substrates required

There are two assay formats available: 1. Fluorescence anisotropy-based (ARC-Fluo Kinase Assay)^[2] and 2. Time-gated luminescence-based (ARC-Lum Kinase Assay)^[3]

Figure 2: Labeled ARC- probes bind to both the substrate- and ATP binding site of protein kinases (PK) from the AGC group with high affinity. The addition of a competitive inhibitor leads to the displacement of the labeled ARC- probe from its complex. The final read-out (fluorescence anisotropy- or time-gated luminescence-based) is determined by the type of reporter group and overall design of the ARC-probe.

Adenosine triphosphate (ATP)-affinity chromatography is a powerful tool

• to achieve high-purity kinase preparations^[5] (Fig. 3A)
• to perform kinase pull-down experiments from crude cell lysates^[6] (Fig. 3B).

Jena Bioscience therefore offers a large selection of ready-to-use ATP-Agaroses

• with different ATP immobilization positions (base, ribose or phosphate moiety)
• in both bulk and column formats

Figure 3: ATP-Agaroses are suitable for both high-purity kinase preparation (A) and kinase pull-down experiments from crude cell lysates (B).
A) ΔN85 c-Src expressed in High 5 insect cells has been purified in a 2-step procedure (1^st Anion exchange chromatography, 2^nd ATP-affinity chromatography). Purity was subsequently checked by SDS-PAGE. 1: clarified lysate, 2: ATP-depleted lysate, 3: Lysate after Q-Sepharose anion exchange chromatography, 4: Purified c-Src after ATP-affinity chromatography with γ-Aminophenyl-ATP-Agarose (10-atom carbon spacer) (AC-101)^[5].
B) 3xFlag-tagged Leucine-Rich Repeat Kinases 1 and 2 (LRRK1 and LRRK2) were pulled-down from crude cell lysates of stably expressed HEK293T cells. The kinase identity was confirmed by subsequent SDS-PAGE and Western blotting with an FLAG antibody. AC-129: N⁶-Aminohexyl-ATP-Agarose, AC-127: 8-Aminohexyl-ATP-Agarose, AC-101: γ-Aminophenyl-ATP-Agarose (10-atom carbon spacer), AC-131: 2'/3'-EDA-ATP-Agarose, Mock beads: blank agarose beads (AC-001)^[6].

[1] Lee et al. (2009) Synthesis and reactivity of novel γ-phosphate modified ATP analogues. Bioorg. Med. Chem. Lett. 19:3804.
[2] Vaasa et al. (2009) High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK. Analytical Biochemistry 385(1):85.
[3] Enkvist et al. (2011) Protein-Induced Long Lifetime Luminescence of Nonmetal Probes. ACS chemical biology 6:1052.
[4] Uri et al. (2012) Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors. Biochimica et Biophysica Acta 1804:541.
[5] Jeansonne et al. (2006) A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src. Protein expression and purification 46:240.
[6] Civiero et al. (2012) Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers. PLOS One 7(8):e43472.