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HaloTag® Technology: Focus on Imaging Technical Manual

Instructions for Use of Product(s)
G2991, G3221, G1001, G1002, G2801, G2802, G8251, G8252, G8272, G8273, G8581, G8582, G9281, G8471, G8472, G9211, P1691, P6751, P6711, P6741, P6771, GA1110, GA1111, GA1120, GA1121
Literature # TM260

The HaloTag® Interchangeable Labeling Technology is a novel tool for imaging live or fixed mammalian cells that express the HaloTag® protein or protein fusions, analyzing posttranslational modification of labeled fusion proteins, and isolating proteins and protein complexes. The technology is based on efficient formation of a covalent bond between a specially designed reporter protein encoded by the HaloTag® pHT2 Vector and a specific ligand in living cells, in solution or on a solid support. The HaloTag® pHT2 Vector contains the open reading frame for a genetically engineered derivative of a hydrolase gene. This protein is not endogenous to mammalian cells.
The ligands carry a variety of functionalities, including fluorescent labels, affinity handles and attachments to a solid phase. The covalent bond forms rapidly under general physiological conditions and is highly specific and essentially irreversible, yielding a complex that is stable even under stringent conditions. The open architecture of the technology enables use of different ligands. We currently offer ligands that can readily cross the cell membrane with blue, red or green fluorophores or biotin. We also offer the cell-impermeant HaloTag® Alexa Fluor® 488 Ligand for labeling cell-surface HaloTag® protein fusions. Additional ligands will be offered to expand the range of applications, as well as solid supports for direct capture of the HaloTag® protein or protein fusions.

Summary of Changes, 10/18 Revision
The following changes were made to the 10/18 revision of this document:
1. Two Janelia Fluor® HaloTag® Ligands were added, including updates to Section 2, Figure 2, Section 4.C (protocol) and a new Figure 6, with subsequent renumbering of the figures following Figure 6. The ligands are Janelia Fluor® 549 HaloTag® Ligand and Janelia Fluor® 646 HaloTag® Ligand.

Printed in USA. Revised 10/18.

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