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Halotag tetramethylrhodamine tmr ligand

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The HaloTag-tetramethylrhodamine (TMR) ligand is a fluorescent dye that binds specifically to the HaloTag protein. The HaloTag is a genetically encoded reporter protein that can be fused to a protein of interest, allowing the visualization and tracking of the protein within living cells.

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Lab products found in correlation

Fla 3000g, by Fujifilm (2 mentions) Nap 5 desalting column, by GE Healthcare (2 mentions) Blue nativepage, by Thermo Fisher Scientific (1 mentions) Halotag succinimidyl ester o4 ligand, by Promega (1 mentions)

Spelling variants of this product

HaloTag TMR ligand (67 citations) HaloTag ligand (16 citations) TMR ligand (5 citations) HaloTag tetramethylrhodamine (TMR; (2 citations)

4 protocols using halotag tetramethylrhodamine tmr ligand

1

Visualizing HaloTag-Labeled Proteins

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The protein of interest (POI) was cloned into HaloTag (HT) vector to establish a HT-POI vector. Cells (1 million cells in 6-well plates) were transfected with 0.5 mg of HT-POI. After culturing them for 40 hours, cells were first incubated with 5 μM HaloTag-tetramethylrhodamine (TMR) ligand (Promega) for 15 min to allow for pulse labeling of the HT-POI and then washed by PBS twice. After that, the cells were suspended in lysis buffer. The whole cell extract was subjected to SDS–PAGE. The TMR-labeled HT-POI was visualized with a fluoro-image analyzer FLA-3000G (FUJI FILM, Tokyo, Japan).
Zhang J., Gan Y., Li H., Yin J., He X., Lin L., Xu S., Fang Z., Kim B.W., Gao L., Ding L., Zhang E., Ma X., Li J., Li L., Xu Y., Horne D., Xu R., Yu H., Gu Y, & Huang W. (2022). Inhibition of the CDK2 and Cyclin A complex leads to autophagic degradation of CDK2 in cancer cells. Nature Communications, 13, 2835.
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2

Fluorescent Protein Labeling Protocol

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Purified proteins containing the HT7 tag were fluorescently labeled for cell-based binding assays as described (Tukachinsky et al., 2016 ). Briefly, proteins were mixed with five-fold molar excess of HaloTag tetramethylrhodamine (TMR) ligand (Promega). After 1-h incubation at room temperature, a NAP-5 desalting column (GE Healthcare) was used to separate labeled protein from unincorporated dye.
Huang P., Wierbowski B.M., Lian T., Chan C., García-Linares S., Jiang J, & Salic A. (2022). Structural basis for catalyzed assembly of the Sonic hedgehog–Patched1 signaling complex. Developmental cell, 57(5), 670-685.e8.
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3

Visualizing Protein Expression with HaloTag

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The protein of interesting (POI) was cloned into HaloTag (HT) vector to establish HT-POI vector. Cells (1 × 106 cells in 6-well plates) were transfected with 0.5 μg of HT-POI, and after cultured for 40 hr, cells were incubated with 5 μM HaloTag-tetramethylrhodamine (TMR) ligand (Promega) for 15 min to allow the pulse labeling of HT-POI, and washed twice with PBS. After the incubation for the indicated time, the cells were washed twice with PBS and suspended in lysis buffer. The whole cell extract was subjected to SDS-PAGE, and the TMR-labeled HT-POI was visualized with a fluoro-image analyzer FLA-3000G (FUJI FILM, Tokyo, Japan).
Gu Y., Zhang J., Ma X., Kim B.W., Wang H., Li J., Pan Y., Xu Y., Ding L., Yang L., Guo C., Wu X., Wu J., Wu K., Gan X., Li G., Li L., Forman S.J., Chan W.C., Xu R, & Huang W. (2017). Stabilization of the c-Myc protein by CAMKIIγ promotes T cell lymphoma. Cancer cell, 32(1), 115-128.e7.
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4

Fluorescent Labeling of HT7 Fusion Proteins

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Purified HT7 fusion proteins were fluorescently labeled as described (Tukachinsky et al., 2016 (link)). Briefly, proteins were incubated for 1 h at room temperature with a five-fold molar excess of HaloTag tetramethylrhodamine (TMR) ligand (Promega). The labeled protein was separated from excess HaloTag ligand using a NAP-5 desalting column (GE Healthcare). For Blue Native PAGE gel shift experiments, 70kDa amino dextran (Thermo) was reacted with a two-fold molar excess of HaloTag succinimidyl ester (O4) ligand (Promega), according to the manufacturer’s instructions. The resulting HaloTag dextran ligand was precipitated by addition of 3 volumes of ethanol. The precipitate was collected by centrifugation, washed with ethanol, dried, resuspended in water and further purified on a NAP-5 desalting column. HaloTag dextran ligand was reacted with purified HPC-HT7::GAS1-Ecto, for 1 h at room temperature. Dextran-conjugated GAS1-Ecto species were purified on a Superdex 200 10/300 GL size-exclusion column.
Wierbowski B.M., Petrov K., Aravena L., Gu G., Xu Y, & Salic A. (2020). Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand. Developmental cell, 55(4), 450-467.e8.
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