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| | | | SNAP-Surface™ Alexa Fluor® 647 | | | |
Description:
SNAP-Surface™ Alexa Fluor® 647 is a photostable fluorescent substrate that can be used to label SNAP-tag® fusion proteins in solution or on the surface of living cells. This cell impermeable substrate is based on Alexa Fluor® 647 and is suitable for standard 635 nm or 650 nm diode laser excitation. It has an excitation maximum at 652 nm and emission maximum at 670 nm. This package includes 50 nmol of SNAP-Surface Alexa Fluor® 647 substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution.
The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a polypeptide based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivates of benzyl purines and benzyl pyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.
There are two steps to using this system: sub-cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Expression of SNAP-tag fusion proteins is described in the documentation supplied with SNAP-tag plasmids. The labeling of the fusion proteins with the SNAP-tag substrate is described below.
Materials Required but not Supplied:
Cells expressing SNAP-Fusion proteins. Proteins of interest can be expressed with the SNAP-tag as either an N-or a C-terminal fusion, but note that the tag needs to be exposed to the extracellular surface of the plasma membrane for labeling with SNAP-Surface Alexa Fluor® 647.
Tissue culture materials and media
Transfection reagents
Fluorescence microscope with suitable filter set
DMSO
Live CHO-K1 cells transiently transfected with pSNAPm-ADRβ2. Cells were labeled with SNAP-Surface Alexa Fluor® 647 (red) for 10-15 minutes and counterstained with Hoechst 33342 (blue).
Figure 1. Excitation (dotted line) and emission (full line) spectra of SNAP-Surface Alexa Fluor® 647 coupled to SNAP-tag in buffer at pH 7.5
Reaction & Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- Storage:
SNAP-Surface Alexa Fluor® 647 should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate should be stable for at least two years dry or 3 months dissolved in DMSO.
Protocols
Protocols for SNAP-Surface™ Alexa Fluor® 647
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Purity and characterization::
Purity of SNAP-Surface Alexa Fluor® 647 was determined to be 99% by HPLC analysis. Molecular weight [M]- was determined by MS to be 1109.3.
In vitro protein labeling::
Reaction of SNAP-Surface Alexa Fluor® 647 (10 µM) with purified SNAP-tag protein (5µM) in vitro, followed by mass spec analysis, indicated an efficiency of labeling of 90%.
Cellular protein labeling::
Cells transfected with SNAP-tag vectors expressing ADRβ2 (cell surface) were labeled with 5 µM SNAP-Surface Alexa Fluor® 647 for 30 minutes, and visualized by confocal microscopy. Only surface target was efficiently labeled.
Legal
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road Ipswich, MA 01938. For detailed information, see www.neb.com/cia/legal/.
This product is provided under an agreement between Life Technologies Corporation and New England Biolabs, and the manufacture, use, sale or import of this product is subject to one or more US patents and corresponding non-US equivalents or other proprietary rights, owned or controlled by Life Technologies Corporation or its affiliates. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product only in research for either in vitro or in vivo directed covalent labeling of proteins conducted by the buyer (whether the buyer is an academic or for-profit entity), according to the product instructions. The buyer must not use the product or its components (1) in manufacturing; (2) to provide a service, information, or data; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, Cellular Analysis Business Unit Development, 29851 Willow Creek Road, Eugene, OR 97402, Tel: (541) 465-8300. Fax: (541) 335-0354.
Alexa-Fluor and Oregon Green are trademarks owned by Life Technologies Corporation
SNAP-Surface and SNAP-Cell are trademarks owned by New England Biolabs, Inc.
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