Citations Search

Search Within Results

Need Assistance

Sort By:

J. Clin. Endocrinol. Metab. 94, 2650-2657. Posttranscriptional Regulation of CDC25A by BOLL Is a Conserved Fertility Mechanism Essential for Human Spermatogenesis 2009

Yung Ming Lin, Chia Ling Chung, and Yu Sheng Cheng

Notes: Human boule (BOLL) gene was subcloned into the HaloTag® pHT2 Vector and transformed into JM109 and HeLa cells. (4060)

Expand Full Notes »

Products

Anal. Biochem. 392, 45-53. Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios. 2009

Hurst, R., Hook, B., Slater, M.R., Hatrnett, J., Storts, D.R., and Nath, N.

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)35S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Expand Full Notes »

Methods in Mol. Biol. 577, 121-131. Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins 2009

Kei Yamaguchi, Shinichi Inoue, Osama Ohara and Takahiro Nagase

Notes: The authors used the Halotag® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 106, 5669-5674. Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin. 2009

Kardon, J.R., Reck-Peterson, S.L. and Vale, R.D.

Notes: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence microscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

Expand Full Notes »

Mol. Biosyst. 4, 59-65. A general system for evaluating the efficiency of chromophore-assisted light inactivation (CALI) of proteins reveals Ru(II) tris-bipyridyl as an unusually efficient "warhead". 2008

Lee, J., Yu, P., Xiao, X. and Kodadek, T.

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

Expand Full Notes »

Science 319, 294. Arabidopsis CLV3 peptide directly binds CLV1 ectodomain. 2008

Ogawa, M., Shinohara, H., Sakagami, Y. and Matsubayashi, Y.

Notes: The authors of this study investigated the role of the genes CLV1 and CLV3 stem cell renewal and differentiation in self-renewing shoot apical meristem (SAM) in Arabidopsis. CLV1 encodes a receptor kinase that is a negative regulator of cell growth, and therefore cannot be overexpressed in plant cells. CLV3, which encodes a protein that is processed to a 12-amino acid peptide, is believed to be a ligand for CLV1. In this study, the authors made a construct in which the CLV1 kinase domain was replaced with the HaloTag™ protein and expressed the fusion protein in tobacco BY-2 cells. The fusion protein was detected using the HaloTag™ TMR ligand. They next incubated membrane extracts from wildtype and transgenic BY-2 cells (expressing the HaloTag™-CLV1 fusion) with tritium-labeled CLV3. The transgenic cells showed significantly higher CLV3 binding than wildtype, and the authors show that CLV3 specifically labels a 130-kd band that corresponds to the HaloTag™-CLV1 fusion protein, indicating that CLV3 binds the ectodomain of CLV1. (3763)

Expand Full Notes »

DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Expand Full Notes »

ACS Chemical Biology 3, 373–382. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis 2008

Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V.

Notes: The authors of this study describe a reporter gene system that allows researchers to create one genetic construct that can be used for a variety of studies including imaging and protein immobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters. (3925)

Expand Full Notes »

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 possesses 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)

Expand Full Notes »

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)

Expand Full Notes »

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)

Expand Full Notes »

Products

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-permeant and impermeant ligands and followed spatial separation of plasma membrane and internal pools of the integrin-HaloTag® protein. (3912)

Expand Full Notes »

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)

Expand Full Notes »

J. Biochem. Biophys. Methods 70, 679-682. Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ 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)

Expand Full Notes »

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)

Expand Full Notes »

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)

Expand Full Notes »

Products

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)

Expand Full Notes »

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)

Expand Full Notes »