SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.
There are several variables affecting the observed results such as focus, laser intensity, equipment capabilities, lens used, etc. The photostability of the fluorophore determines how quickly the fluorescence must be captured before fading.
This method of protein labeling is versatile and suitable for many applications. SNAP-tag and CLIP-tag technologies for the specific labeling of fusion proteins with synthetic probes enable various aspects of protein function, including a variety of dynamic processes in live cells and in cell lysates. SNAP-, CLIP-, MCP- and ACP-tag technologies provide simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of forming a covalent linkage to a variety of functional groups, including fluorophores, biotin, or beads. The protein of interest must be cloned and expressed only once and then the fusion can be used with a variety of fluorescent substrates for numerous downstream applications such as simultaneous protein labeling inside live cells, protein localization and translocation, pulse-chase experiments, receptor internalization studies, selective cell surface labeling, protein pull down assays, protein detection in SDS-PAGE, flow cytometry, high-throughput binding assays in microtiter plates, biosensor interaction experiments, and FRET-based binding assays.
The authors have nothing to disclose.
Kai Johnsson
Ivan Correa
Andreas Brecht
Katrin Müentener
Chris Noren
Luo Sun
Ming Xu
Theodore Davis
Salvatore Russello
Aihua Zhang
Inca Ghosh
Elissa Maunus
Nicholas Labarthe
James MacFarland
John Buswell
Brenda Desmond
James Ellard
Don Comb
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
CHO-K1 Cells | ATCC | CCL-61 | ||
Serum free F-12K | ||||
Lab-Tek II Chambered Coverglass with #1.5 borosilicate coverglass chambers | Nalgene Nunc Int. | 155409 | Lot: 100808-8-0 | |
DMEM | ||||
Hoechst 33342, trihydrochloride, trihydrate (10 mg/ml solution in water, 16.2 mM solution) | Invitrogen Molecular Probes | H3570 | 470519 | |
Zeiss Apotome Axiovert 200M fluorescent microscope | Zeiss | |||
Transpass V | New England Biolabs | M2561S | 0030709 | |
Transpass D2 | New England Biolabs | M2554S | 0060802 | |
SNAP-Cell TMR Star | New England Biolabs | S9105S | 0010811 | |
pSNAP-ADBR2 Control Plasmid | New England Biolabs | N9178S | 0010811 | |
pSNAP-H2B Control Plasmid | New England Biolabs | N9179S | 0010811 |