Protein labelling technology consists of a single vector encoding a specially designed protein which can form a covalent link with one of three ligands carrying different functionalities
Promega has introduced a new protein labelling technology that allows imaging and/or protein capture in either live or fixed mammalian cells.
HaloTag interchangeable labelling technology consists of the single HaloTag pHT2 vector encoding a specially designed protein which can form a covalent link with one of three HaloTag ligands carrying different functionalities.
It obviates the need to design and clone new expression constructs for different applications.
The highly specific bond between reporter protein and ligand forms rapidly under physiological conditions and is irreversible.
The technology can be used on living or fixed cells, either in solution or on a solid support, and is therefore applicable for protein localisation, protein trafficking, protein capture and cell-to-gel analysis.
The HaloTag pHT2 Vector encodes the HaloTag protein, which is a genetically engineered derivative of haloalkane dehalogenase.
A gene of interest can be cloned into the vector so that either N- or C- terminal fusion proteins can be made.
The HaloTag ligands bind to the HaloTag portion of the fusion protein.
The three ligands currently available are:.
HaloTag TMR ligand - containing tetramethyl rhodamine (TMR) and suitable for fluorescence measurements at 555Ex/585Em (in the red area of the spectrum).
HaloTag diAcFAM ligand - containing a non-fluorescent derivative of fluorescein, which is metabolised to brightly fluorescent FAM; measurements can be made at 494Ex/526Em (in the green area of the spectrum).
HaloTag biotin ligand - containing biotin and suitable for use as an affinity tag to capture the protein of interest.
The ability of the ligand to cross cell membranes efficiently allows the technology to be used with live cells.
It can also be used alongside GFP for multiple protein visualisation but in contrast to GFP, the strength of the covalent bond in the HaloTag system allows for stable imaging of the fusion protein in fixed cells.
The bond is also stable in denaturing conditions required for applications such as SDS-Page or mass spectrometry.
