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ACS Chemical Biology 11, 2608–17. Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag 2016

Friedman Ohana, R., Levin, S., Wood, M.G., Zimmerman, K., Dart, M.L., Schwinn, M.K., Kirkland, T.A., Hurst, R., Uyeda, H.T., Encell, L.P. and Wood, K.V.

Notes: In this publication, HaloTag® technology was used to identify the protein targets of specific compounds. The tested compounds, kinase inhibitors ibrutinib and BIRB796, were modified with a chloroalkane tag, introduced into cells and after incubation, the cells were lysed. The cellular proteins that interacted with the compounds were captured onto resin with immobilized HaloTag® protein and palladium added to chemically cleave the bound protein from the tagged compound. The protein targets were then identified using mass spectrometry. (4941)

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Cell 154, 541–555. KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors 2013

Black, J.C., Manning, A.L., Van Rechem, C., Kim, J., Ladd, B., Cho, J., Pineda, C.M., Murphy, N., Daniels, D.L., Montagna, C., Lewis, P.W., Glass, K., Allis, C.D., Dyson, N.J., Getz, G. and Whetstine, J.R.

Notes: The HaloTag® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

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J. Clin. Endocrinol. Metab. 94, 2594-2601. KLF15 Is a transcriptional regulator of the human 17beta-hydroxysteroid dehydrogenase type 5 gene. A potential link between regulation of testosterone production and fat stores in women 2009

Du, X., Rosenfield, R. and Qin, K.

Notes: The authors used a HaloTag® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

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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)

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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 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)

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