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J. Biol. Chem. 283, 11575-11585. Identification of ubiquitin ligase activity of RBCK1 and its inhibition by splice variant RBCK2 and protein kinase Cβ. 2008

Tatematsu, K., Yoshimoto, N., Okajima, T., Tanizawa, K., and Kuroda, S.

Notes: RBCK1 is a RING-IBR (ring in between ring fingers) protein previously shown to have transcriptional activity and to bind protein Kinase Cβ. This paper demonstrates that RBCK1 also posseses ubiquitin ligase E3 activity. Both FLAG-tag and HaloTag® labeled proteins were used to demonstrate this activity. To demonstrate the interaction between RBCK1 and ubiquitinated proteins, HaloTag®-ubiquitin and FLAG-RBCK1 were coexpressed in HEK293 cells in the presence or absence of a proteasome inhibitor. The anti-FLAG immunoprecipitates isolated from these cells were analyzed by SDS-PAGE and Western blotting using anti-HaloTag® and anti-FLAG antibodies and self-ubiquitination of RBCK1 was demonstrated. RBCK2, a splice variant of RBCK1 lacking the RING domain showed no self ubiquitination activity but was demonstrated to interact with RBCK1 in vivo and in vitro, and to inhibit the self-ubiquitination activity of RBCK1 in a FLAG-tag assay. Pulse-chase experiments, using HEK293 cells expressing HaloTag®-RBCK1 with or without RBCK2 and treated with HaloTag®-TMR ligand, were used to show that the half-life of RBCK1 was extended by overexpression of RBCK2. (3874)

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J. Proteome Res. 7(10), 4475-82. Improving protein array performance: focus on washing and storage conditions. 2008

Nath, N., Hurst, R., Hook, B., Meisenheimer, P., Zhao, K.Q., Nassif, N., Bulleit, R.F., and Storts, D.R.

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4oC but slides stored at -20oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

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Appl. Environ. Microbiol. 73, 4234-4242. Rapid engineering of bacterial reporter gene fusions by using Red recombination. 2008

Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M.

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag® reporter using the HaloTag® coding region from the pHT2 promoter. (3924)

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BMC Cell Biology 9:17, doi:10.1186/1471-2121/9/17. Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter. 2008

Svendsen, S., Zimprich, C., McDougall, M.G., Klaubert, D.H., and Los, G.V.

Notes: This paper demonstrates use of HaloTag® technology to study expression, trafficking and translocation of an integrin-HaloTag® fusion protein. The authors fused the Halotag reporter protein to truncated integrin. They then labeled live cells with different cell-permaent and impermeant ligands and followed spatial separation of plasma membrane and internal pools of the integrin-HaloTag® protein. (3912)

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Plant J. 52, 175–184. Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing. 2007

Shinohara, H. and Matsubayashi, Y.

Notes: The authors were looking for a method for ligand fishing experiments with plant receptor-like kinases (RLKs). The strategy chosen, functional immobilization on microbeads, used phytosulfokine (PSK) and its carrot cell receptor (DcPSKR1) as the test ligand-receptor pair. DcPSKR1 was cloned into the pHT2 HaloTag® Vector, adding the HaloTag® gene to the C terminus of DcPSKR1 or replacing the DcPSKR1 kinase domain. These constructs were transfected into BY-2 cells and expression confirmed by immunoblotting the membrane fraction and staining with DcPSKR1 and Anti-HaloTag antibodies. To confirm activity of the individual proteins, membrane fractions of BY-2 cells expressing the DcPSKR1-HaloTag® fusion proteins were tested for PSK binding activity to then HaloTag® binding activity confirmed using the HaloTag® TMR Ligand. The DcPSKR1-ΔKD-Halo protein was immobilized on HaloLink™ Resin and the Kd measured. The binding of PSK to the immobilized DcPSKR1-ΔKD-Halo protein was visualized by using fluorescently labeled Alexa488-PSK. Columns of HaloLink™ Resin with bound DcPSKR1-ΔKD-Halo protein were used to bind PSK ligands at physiological concentration, elute the ligands with a high-salt buffer and analyzed on LC-MS. (3946)

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J. Biochem. Biophys. Methods 70, 679-682. Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTagtrade Technology. 2007

Hata, T. and Nakayama, M.

Notes: This paper outlines a method for the affinity purification of polyclonal antisera using the HaloTag® Technology. The protein of interest was expressed in E. coli as a HaloTag® fusion protein. Bacterial cell lysate (100µl) was then mixed with 50µl of HaloLink™ Resin (50% suspension) for 1 hour at room temperature. After washing, 100µl of immune serum (1:10 dilution) was added and the mixture incubated for 1 hour at 4°C. The resin was then washed three times with a high-salt buffer to remove nonspecific antibodies prior to elution of specifically bound antibodies using 100mM glycine (pH 2.5). The authors discuss the advantages of this single-tube method compared to other methods of antibody purification. (3623)

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J. Biol. Chem. 282, 13059-13072. XAF1 mediates tumor necrosis factor-α-induced apoptosis and X-linked inhibitor of apoptosis cleavage by acting through the mitochondrial pathway 2007

Straszewshi-Chavez, A., Visintin, I.P., Karassina, N., Los, G., Liston, P., Halaban, R., Fadiel, A. and Mor, G.

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

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Angewandte Chemie International Edition 45, 4536-4940. HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots. 2006

Zhang, Y., So, M.-K., Loening, A.M., Yao, H., Gambhir, S.S., and Rao, J.

Notes: This paper describes work to use the HaloTag™ Interchangeable Protein Labeling Technology for the specific conjugation of proteins to quantum dots. The authors used the pHT2 vector to create a Renilla luciferase (Luc 8)/HaloTag™ Protein fusion that contained a 6X polyhistidine tag. The fusion protein was purified over a nickel affinity resin. They synthesized a HaloTag™ ligand for conjugation to the quantum dots. The ligand was designed to orient the ligand away from the quantum dot surface to minimize steric hindrance between the HaloTag™ fusion protein and the quantum dots upon interaction of the fusion protein with the ligand. The authors used BRET emission to evaluate conjugation of the HaloTag™ fusion protein to the quantum dots. They conclude that using this technology, they were able to conjugate a bioluminescent protein to quantum dots, creating self-illuminating quantum dots. Furthermore, they suggest that the mild conjugation conditions used may allow in vivo labeling of proteins or cells using quantum dots. (3490)

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J. Exp. Bot. July, epub ahead of print. HaloTag™: A new versatile reporter gene system in plant cells. 2006

Lang, C., Schulze, J., Mendel, R-R. and Hänsch, R.

Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM®-T Easy® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-35S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

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Cell 126, 335-348. Single-molecule analysis of dynein processivity and stepping behavior. 2006

Reck-Peterson, S.L., Yidiz, A., Carter, A.P., Gennerich, A., Zhang, N. and Vale, R.D.

Notes: HaloTag™ Interchangeable Labeling Technology was used to specifically label engineered dynein produced in Saccharomyces cerevisiae. The HaloTag™ Protein was added in-frame with either the 5´ or 3´ end of the coding sequence of various engineered dynein molecules. The authors report being able to label the dynein in specific locations using fluorescent dyes or quantum dots. HaloTag™ TMR Ligand was used to covalently label dynein to directly visualize dynein motor movement using total internal reflection fluorescence microscopy. HaloTag™ Biotin Ligand was used to label dynein with streptavidin quantum dots. (3504)

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