Division of Chemical Biology, New England Biolabs
Provost, C. R., Sun, L. Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging. J. Vis. Exp. (39), e1876, doi:10.3791/1876 (2010).
Plating CHO-K1 Cells:
Transient Transfection of CHO-K1 Cells with pSNAP-ADRB2 and pSNAP-H2B:
|plasmid||number of reactions||μl of serum-free medium||μl of TransPass D2||μl of TransPass V||μl of plasmid DNA||μl of TransPass D2/reaction||μl of TransPass V/reaction||ng of DNA/ reaction||DNA conc. (ng/μl)||μl of plasmid DNA/ reaction|
SNAP-Cell TMR Star Labeling of CHO-K1 Cells Transiently Transfected with pSNAP-ADRB2 and pSNAP-H2B :
Figure 1. Here are some representative results of fluorescent imaging using standard fluorescent microscopy of live COS-7 cells that are expressing SNAP-tag fusions. This first image shows live COS-7 cells expressing pSNAP-H2B (nuclear histone) labeled with SNAP-Cell TMR-Star. The pSNAP-H2B construct was generated using the pSNAP-tag(m) vector. The cells were labeled with SNAP-Cell TMR-Star for 30 minutes and counterstained with Hoechst 33342 (blue) for nuclei. The pink fluorescence demonstrates the overlay of red fluorescence where histone protein is present in addition to the blue fluorescence indicating the nucleus. The expression of the H2B protein is clearly and easily identified.
Figures 2 and 3. These next two images show live COS-7 cells transiently transfected with pSNAP-ADRβ2.
Cells were labeled with SNAP-Cell TMR-Star (red) and counterstained with Hoechst 33342 (blue). The pSNAP-ADRβ2 construct was generated using the pSNAP-tag(m) vector. The cells were labeled with SNAP-Cell TMR-Star for 30 minutes and counterstained with Hoechst 33342 (blue) for nuclei. The red fluorescence indicates the presence of cell surface receptor protein ADRB2 while blue fluorescence identifies the nucleus.
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 are employed by New England Biolabs that produces the reagents and instruments used in this article.
|Serum free F-12K|
|Lab-Tek II Chambered Coverglass with #1.5 borosilicate coverglass chambers||Nalge Nunc international||155409||Lot: 100808-8-0|
|Hoechst 33342, trihydrochloride, trihydrate (10 mg/ml solution in water, 16.2 mM solution)||Invitrogen||H3570||470519|
|Zeiss Apotome Axiovert 200M fluorescent microscope||Carl Zeiss, Inc.|
|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|