Product Class: Other

CLIP-Cell™ Block

  • Catalog # S9220 was discontinued on December 15, 2022

Product Introduction

CLIP-Cell™ Block (bromothenylcytosine, BTC) is a non-fluorescent compound that blocks the reactivity of the CLIP-tag™ in vitro and inside or on the surface of living cells.

  • It is cell permeable
  • It can be used to generate inactive controls in live cell labeling experiments performed with CLIP-tag fusion proteins
  • It reacts with CLIP-tag irreversibly, inactivating it for subsequent labeling steps
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Product Information

Description

The CLIP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a protein tag based on human O6-alkylguanine-DNAalkyltransferase (hAGT). CLIP-tag substrates are derivatives of benzylcytosine (BC). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether bond.  Although CLIP-tag is based on the same protein as SNAP-tag®, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag can be used for orthogonal and complementary labeling of two proteins simultaneously in the same cells. 


There are two steps to using this system: subcloning and expression of the protein of interest as a CLIP-tag fusion, and labeling of the fusion with the CLIP-tag substrate of choice. The use of BTC during the labeling of fusion proteins with CLIP-Cell substrates is described in this document.

Figure 1: Structure of CLIP-Cell Block (MW 286.1) Figure 1: Structure of CLIP-Cell Block (MW 286.1)
This product is related to the following categories:

Properties & Usage

Materials Required but not Supplied

  • Cells expressing CLIP-tag fusion proteins 
  • Tissue culture materials and media 
  • Transfection reagents 
  • Fluorescence microscope with suitable filter set 
  • DMSO

Product Notes

  1. Storage: CLIP-Cell Block should be stored at -20°C (long term) or at 4°C (short term). With proper storage at -20°C, CLIP-Cell Block is stable for at least three years dry or for three months when dissolved in DMSO.
  2. For troubleshooting please refer to the instructions supplied with CLIP-Cell products as appropriate.
  3. Please note that there is a constant turnover and resynthesis of proteins in the cell. After having blocked all existing CLIP-tag fusion proteins within the cell, new CLIP-tag fusion protein molecules may be synthesized in the meantime and may get labeled during incubation with a fluorescent CLIP-tag substrate. This will give the impression that the blocking was ineffective. In order to minimize these effects of protein synthesis and protein transport, cells may have to be treated with cycloheximide and incubation with the fluorescent CLIP-tag substrate may have to be performed at 4°C.

Protocols, Manuals & Usage

Protocols

  1. Use with CLIP-tag substrates (S9220)
  2. Labeling of Proteins in vitro (S9220)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the SNAP-tag®?
  2. How does it work?
  3. How specific is the binding of substrate to the SNAP-tag®?
  4. How does SNAP-tag labeling differ from using GFP fusion proteins?
  5. What linker type and length would you recommend?
  6. Can I clone my protein as a fusion to the N- or C-terminus of the tags?
  7. What is the smallest peptide and biggest protein you have cloned as SNAP-tag fusions?
  8. What is the solubility of SNAP-tag in insect and bacterial expression systems?
  9. What competent cell strains does NEB suggest for expression in E. coli?
  10. What competent cell E. coli strains are suitable for propagating SNAP-tag plasmids?
  11. Can SNAP-tag fusions be purified and refolded from inclusion bodies?
  12. Are substrates toxic to cells?
  13. Can cells expressing SNAP-tag be fixed prior to labeling?
  14. Can SNAP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
  15. Can you use SNAP-tag for in vivo FRET?
  16. Can cell-impermeable substrates be microinjected into cells, and how is the excess substrate exported?
  17. Does the SNAP-tag labeling reaction work in yeast?
  18. What happens to the fluorophore upon proteolysis?
  19. What conditions are recommended for SNAP-tag labeling in vitro?
  20. What conditions are incompatible with SNAP-tag labeling in vitro?
  21. Can SNAP-tag fusion proteins be labeled in a cell lysate?
  22. I have a compound that I would like to couple to a BG derivative. Where can I get advice?
  23. What is the difference between SNAP-tag and ACP-tag?
  24. Cellular Imaging and Analysis FAQs

Troubleshooting

Tech Tips

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).

Quality, Safety & Legal

Quality Assurance Statement

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.

Specifications

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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:
Labeling of Fusion Proteins with Synthetic Probes: US 8,227,602; US 8,623,627; and EP 2 049 499.

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