SNAP-Cell® Fluorescein is a green fluorescent substrate that can be used to label SNAP-tag® fusion proteins inside living cells or in vitro. This substrate has limited photostability.
It is a cell-permeable substrate (BG-Fluorescein) based on diacetylfluorescein
Diacetylfuorescein is essentially non-fluorescent, but it becomes fluorescent inside the cell when it is hydrolyzed by non-specific esterases, yielding fluorescein
It is suitable for standard fluorescein filter sets
It has an excitation maximum at 500 nm and an emission maximum at 532 nm
This package contains 50 nmol of SNAP-Cell Fluorescein substrate, sufficient to make 10 ml of a 5 µM SNAP-tag fusion protein labeling solution. This substrate has limited photostability. If this presents a problem, we recommend using SNAP-Cell 505, which has similar spectral characteristics but much greater photostability.
The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzylpurines and benzylchloropyrimidines, In the labeling reaction, the substituted benzyl group of the substrate becomes 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 fusion proteins with the SNAP-tag substrate is described below.
Figure 1:
Live CHO-K1 cells transiently transfected with pSNAP-H2B
Cells were labeled with SNAP-Cell Fluorescein (green) for 15 minutes.Figure 2: Excitation (dotted line) and emission spectra of SNAP-Cell Fluorescein (fluorescent product of SNAP-Cell Fluorescein in cells) coupled to SNAP-tag in buffer at pH 7.5)
Figure 3: Structure of SNAP-Cell Fluorescein (MW 712.7g/mol)
Storage: SNAP-Cell Fluorescein 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 3 years dry or 3 months dissolved in DMSO.
After addition of DMSO, pipette up and down at least 10-20 times and vortex vigorously for at least one full minute to ensure full dissolution of the substrate.
After diluting the substrate in complete medium, thoroughly pipette up and down at least 10 times to help reduce background.
Increasing substrate concentration and/or reaction time usually results in higher background and does not necessarily increase the signal-to-background ratio (SNR).
After the wash steps, incubate the intracellular labeling samples at 37°C, 5% CO2 for 30 extra minutes to allow any unincorporated fluorophore to diffuse out of the cells.
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Licenses
Cellular Imaging and Analysis (i.e., SNAP and CLIP products)
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT 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 our Terms of Use .
These patents and patent applications are owned by Covalys, or owned by the Ecole Polytechnique Fédérale de Lausanne (EPFL) and exclusively licensed to Covalys and NEB.
The products and/or their use may be covered by one or more of the following patents and patent applications: Methods for Using O6-Alkylguanine-DNA-Alkyltransferases: US 7,939,284; US 8,367,361; EP 1 410 023; EP 2 037 271; EP 1 696 234; EP 2 211 177; JP 4195815; JP 4226053; CN 1295510; CN 1975422; and SG 100125.
Substrates for O6-Alkylguanine-DNA Alkyltransferase: US 7,799,524; and JP 4976651.
Mutants of O6-Alkylguanine-DNA-Alkyltransferases: US 7,888,090. EP 1720981 (pending).
Specific Substrates for O6-Alkylguanine-DNA-Alkyltransferases: US 8,163,479. EP 1730298 (pending).
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