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Cellular Imaging and Analysis

New England Biolabs introduces a set of tools for the specific labeling of fusion proteins with synthetic probes that help scientists illuminate various aspects of protein function, including a variety of dynamic processes in live cells and in cell lysates. Developed by scientists at the Ecole Polytechnique Federale de Lausanne, these "SNAP-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. > more information on the science behind these tags.

 


A fusion protein is created with the protein of interest (blue) and SNAP-tag™ (gold). Addition of fluorogenic substrate (green) results in the fluorophore being covalently attached to the fusion protein.

 

Starter Kits: To help customers begin to use SNAP-tag technologies to visualize proteins in live and fixed cells, NEB is introducing 5 Starter Kits: SNAP-Cell Starter Kit, SNAP-Surface Starter Kit, CLIP-Cell Starter Kit, CLIP-Surface Starter Kit and ACP-Surface Starter Kit (coming soon). Each Starter Kit contains a plasmid encoding the tag of interest and two cell permeable or non-cell permeable fluorescent labels. A positive control plasmid encoding a tagged protein with a well-characterized sub-cellular localization is also included (e.g. membrane, nucleus, etc). Lastly, where available, a negative control "blocking-agent" is included that interacts with the tag of interest, but is not fluorescent.

For a detailed outline about how to choose a specific product for your application, please see our Applications Selection Chart. For general information about each product line, see below.

 

SNAP-Cell: SNAP-Cell labels are cell-permeable and uniquely suited to the labeling of SNAP-tag fusion proteins inside living cells, on cell surfaces or in vitro. These labels spread across the visible spectrum, ranging from blue to red. Non-fluorescent cell-permeable blocking agents are also available.
SNAP-Surface: SNAP-Surface labels are non-cell permeable and routinely used to label SNAP-tag fusion proteins on the surface of living cells, in fixed cells or in vitro. These labels are spread across the visible and near-infrared spectrum and include the photostable AlexaFluor® dyes and a variety of other commonly used fluorophores. Non-fluorescent blocking agents are also available. The SNAP-Vista labels can be used to visualize proteins directly in SDS-Page.
CLIP-Cell: CLIP-Cell labels are cell-permeable and uniquely suited to the labeling of CLIP-tag fusion proteins inside living cells, on cell surfaces or in vitro. The CLIP-tag is a derivative of the SNAP-tag that reacts with orthogonal substrates, allowing simultaneous labeling of 2 expressed proteins. The labels spread across the visible spectrum, ranging from blue to red. Non-fluorescent cell-permeable blocking agents are also available.
CLIP-Surface: CLIP-Surface labels are non-cell permeable and routinely used to label CLIP-tag fusion proteins on the surface of living cells, in fixed cells or in vitro. The CLIP-tag is a derivative of the SNAP-tag that reacts with orthogonal substrates, allowing simultaneous labeling of 2 expressed proteins. The labels include fluorophores at commonly used areas of the visible spectrum, like 488, 547 and 647nm. CLIP-Vista Green can be used to visualize proteins directly in SDS-Page.

 

ACP- & MCP-Surface: ACP and MCP-Surface products differ from their SNAP-tag and CLIP-tag counterparts in that they require a specific synthase to become labeled, ACP or SFP Synthase respectively. Labels at 488, 547 and 647 nm are available for this system.

Biotin-tags: For maximal flexibility with existing detection technologies, biotinylated tags are available, including: SNAP-Biotin, CLIP-Biotin and CoA-Biotin.

SNAP-Capture: To enable protein pull-down experiments and proteomic analysis, SNAP-Capture Pull-Down Resins and SNAP-Capture Magnetic Beads are available.

Building blocks: For advanced users with novel probes interested in working with SNAP-tag technologies, a complete line of building blocks are available for linkage of the core benzylguanine (BG) and benzylcytosine (BC) and CoA moieties to activated esters, primary amines and thiol groups.

 

Background Introduction

An ever increasing number and variety of tagging technologies have become popular in various branches of biology and biochemistry. For example, the practice of cell biology without the use of fluorescent proteins is unimaginable today and the purification of recombinant proteins using affinity tags is standard practice.

However, despite the ubiquitous role of fusion proteins in protein science, traditional tags face two major limitations:

1. They are limited to properties that can be genetically encoded. For example, the spectroscopic properties of currently available fluorescent proteins are often inferior to synthetic fluorophores, but such fluorphores cannot be genetically encoded.


2. Traditional tags are tailor-made for a specific application and a single tag is not suited to the study of different facets of protein function. An approach that addresses these shortcomings relies on tags that can be specifically labeled with synthetic probes such as fluorophores or affinity labels.

New England Biolabs is pleased to announce the release of a line of multifunctional protein labeling tools. These include: SNAP-tag, CLIP-tag, ACP-tag, MCP-tag and a complete line of labels that covalently interact with each tag.

The most versatile and best-known of these tags is the SNAP-tag, a 20 kDa mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase that reacts specifically and rapidly with benzylguanine (BG) derivatives, leading to the covalent labeling of SNAP-tag with a synthetic probe. SNAP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of SNAP-tag with BG derivatives is to a large extent independent of the nature of the synthetic probe attached to BG, permitting the labeling of SNAP fusion proteins with a wide variety of synthetic probes.

The second tag introduced by NEB is a sibling of the SNAP-tag, the CLIP-tag. The CLIP-tag was created by engineering the substrate specificity of SNAP-tag, permitting it to react exclusively with benzylcytosine (BC) derivatives rather than BG derivatives. Since SNAP-tag and CLIP-tag react with different substrates, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different synthetic probes in living cells. A central application of the CLIP-tag is dual-labeling of fusion proteins in conjunction with the SNAP-tag.

The third tag is conceptually different from the labeling of SNAP and CLIP fusion proteins as it is based on an enzyme-catalyzed post-translational modification. The protein of interest is fused to an acyl carrier protein (ACP-tag) and the corresponding fusion protein is specifically labeled with CoA derivatives by the phosphopantetheinyl transferases ACP Synthase or Sfp Synthase. An interesting feature of the ACP-tag is its small size of 9 kDa. A mutant of the ACP-tag, called the MCP-tag, is labeled by Sfp Synthase but not by ACP Synthase, permitting the selective labeling of ACP and MCP fusion proteins with different probes in one sample.





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