Halotag7

Manufactured by Promega

The HaloTag7 is a genetically encoded protein tag that can be covalently attached to a Halo ligand. This allows for the detection, purification, and immobilization of proteins tagged with HaloTag7.

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Lab products found in correlation

Pspcas9 bb 2a gfp, by Addgene (1 mentions) Snap tag, by New England Biolabs (1 mentions) Pfc14k halotag cmv flexi vector, by Promega (1 mentions) Pegfp n1 mammalian expression vector, by Takara Bio (1 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Pcdna3.1 mammalian expression vector, by Thermo Fisher Scientific (1 mentions)

3 protocols using halotag7

Generation and Characterization of WIPI and ATG2A Constructs

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DNA fragments encoding WIPI1 (NM_017983.7), WIPI2 (NM_016003.4), WIPI3 (NM_019613.4), WIPI4 (NM_001029896.2), and their variants were inserted into the retroviral plasmids pMRXIP (68 (link)), pMRXIB and pMRXIH together with 3 × FLAG tag or HaloTag7 (N2701, Promega, Madison, WI). DNA fragments encoding ATG2A (NM_015104.3) and its variants were inserted into pMRXIP (68 (link)) together with monomeric ultrastable green fluorescent protein (muGFP). DNA fragments encoding ATG2A (NM_015104.3) and its variants were also inserted into pMRXNo. The pMRXIB, pMRXIH and pMRXNo plasmids were constructed by replacing a puromycin-resistant gene cassette with a blasticidin-resistant gene cassette, a hygromycin B-resistant gene cassette, and an internal ribosomal entry site driven SNAP-tag (N9181S, New England BioLabs, Ipswich, MA), respectively. Mutated or truncated constructs were generated by PCR-mediated site-directed mutagenesis. The pMRXIP-GFP-LC3-RFP, pMRXIB-Halo-mGFP and pMRXNo-Halo-ratLC3B plasmids were constructed as described previously (41 (link),42 (link)).
For the generation of KO cell lines, guide RNAs (gRNAs) were cloned into pSpCas9(BB)-2A-GFP (PX458; a gift from Dr F. Zhang, Broad Institute of Massachusetts Institute of Technology; #48138, Addgene, Watertown, MA).

Shimizu T., Tamura N., Nishimura T., Saito C., Yamamoto H, & Mizushima N. (2023). Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration. Human Molecular Genetics, 32(16), 2623-2637.

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Generation of Plasmids for Stable Expression in HeLa Cells

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Plasmids for stable expression in HeLa cells were generated as follows: DNA fragments encoding monomeric enhanced GFP with A206K mutation (mGFP), monomeric RFP (mRFP), mRuby3 (codon-optimized from Addgene #74252), HaloTag7 (Promega, N2701), or SNAP-tag (New England BioLabs, N9181S) were inserted into the retroviral plasmids pMRX-IP (harboring a puromycin-resistant marker; Kitamura et al., 2003 (link); Saitoh et al., 2003 (link)), pMRX-IB (harboring a blasticidin-resistant marker; Morita et al., 2018 (link)), or pMRX-No (without a drug resistance marker) by the seamless ligation cloning extract (SLiCE) method (Motohashi, 2017 (link)). Then, DNA fragments encoding rat LC3B, the signal sequence of Drosophila BiP (residues 1–18) and KDEL (for pMRX-IB-Halo-mGFP-KDEL), or the presequence of Neurospora crassa Fo-ATPase subunit 9 (residues 1–69; for pMRX-IB-pSu9-Halo-mGFP) were inserted into the pMRX-IP-, pMRX-IB-, pMRX-No-based plasmids by the SLiCE method. mRFP-GFP-LC3B in pMXs was described previously (Jiang et al., 2014 (link)). Primers used in this study are listed in Supplementary file 1. Plasmids containing the Halo constructs used in this study can be found in addgene: 184899 (Halo-LC3), 184901 (Halo-mGFP-rLC3), 184902 (Halo-mGFP), 184904 (Halo-mGFP-KDEL), and 184905 (pSu9-Halo-mGFP).

Yim W.W., Yamamoto H, & Mizushima N. (2022). A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag. eLife, 11, e78923.

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Protein fusion and expression techniques

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The HaloTag7 (Promega) coding sequence was amplified by PCR, and was fused at the C-terminus of mouse mGluR3 with an In-Fusion HD Cloning Kit (Clontech). To quantify the expression of wild type and HaloTag-fused mGluR3 by Western blotting analysis, the epitope sequence of the anti-bovine rhodopsin monoclonal antibody Rho1D4 was also fused at the C-terminus. The cDNAs of mGluR3s were introduced into the pcDNA3.1 mammalian expression vector (Invitrogen). The cDNAs of other GPCRs (ADRB2, HTR2A, HRH1, ADORA2A, FFAR4, CXCR4, F2R, GCGR) were purchased from Promega, and the receptor coding sequences were inserted into pFC14K HaloTag CMV Flexi Vector. The CD86 (M1-R277) coding sequence was amplified by PCR, and inserted into the pEGFP-N1 mammalian expression vector (Clontech), where the coding sequence of EGFP was swapped with that of HaloTag7. The SNAP-tag (NEB) coding sequence was amplified by PCR and inserted in the loop region between L91 and G92 of the mouse Gα o subunit coding cDNA, as reported previously (12) . The SNAP-tagged Gα o subunit coding sequence was inserted into the pFC15A HaloTag CMVd1 Flexi Vector without the HaloTag coding sequence to reduce the expression level for single-molecule imaging. The cDNA of GFP-tagged CLC was constructed as previously reported (45) (link).

Yanagawa M., Hiroshima M., Togashi Y., Abe M., Yamashita T., Shichida Y., Murata M., Ueda M, & Sako Y. (2018). Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors. Science signaling, 11(548).

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