Product Class: Other

CLIP-Biotin

Product Introduction

CLIP-Biotin is suitable for applications such as biotinylation of CLIP-tag™ fusion proteins in living cells for detection with streptavidin fluorophore conjugates or labeling in solution for analysis by SDS-PAGE/Western blot or for capture with streptavidin for binding and interaction studies.

  • It is a cell-permeable substrate (BC-Biotin)
  • It is based on biotin with an amidocaproyl linker
Catalog # Size Concentration
S9221S 50.0 nmol

Product Information

Description

This package contains 50 nmol of CLIP-Biotin substrate, sufficient to make 10 ml of a 5 μM solution for the labeling of CLIP-tag fusion proteins in cells.

CLIP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). 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 link. 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 simultaneous labeling.

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. Expression of CLIP-tag fusion proteins is described in the documentation supplied with CLIP-tag plasmids. The labeling of fusion proteins with CLIP-Biotin is described below.
9147a_thumb
Figure 1: Structure of CLIP-Biotin (MW 569.7 g/mol)
This product is related to the following categories:
Biotin Labels Products,
CLIP-tag™ Substrates Products,
Protein Labeling Products,
This product can be used in the following applications:
CLIP Cell,
In vivo Imaging,
Protein Localization,
Protein Labeling Snap Clip

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Materials Required but not Supplied

  • Cells expressing CLIP-tag proteins 
  • Tissue culture materials and media 
  • Transfection reagents 
  • DMSO

Product Notes

  1. Storage: CLIP-Biotin 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 three years dry or 3 months dissolved in DMSO.

Protocols, Manuals & Usage

Protocols

  1. Cellular Labeling (S9221)
  2. Labeling of Proteins in vitro (S9221)

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Cellular Imaging and Analysis FAQs

Troubleshooting


Troubleshooting for Labeling in vitro 

Solubility
If solubility problems occur with your CLIP-tag fusion protein, we recommend testing a range of pH (pH 5.0–pH 10.0) and ionic strengths. The salt concentration may also need to be optimized for your particular fusion protein (50–250 mM).

Loss of Protein Due to Aggregation or Sticking to Tube
If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The CLIP-tag activity is not affected by this concentration of Tween 20.

Incomplete Labeling
If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Increase the incubation time to two hours total at 25°C or to 24 hours at 4°C; or halve the volume of protein solution labeled. Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the CLIP-tag using CLIP-Vista Green.

If the CLIP-tag fusion has been stored in the absence of DTT or other reducing agent, or has been stored at 4°C for a prolonged period, its activity may be compromised. Include 1 mM DTT in all solutions of the CLIP-tag fusion protein, and store the fusion protein at -20°C.

Using less than the recommended amount of substrate stock solution can significantly slow down the reaction rate.

Loss of Activity of Protein of Interest
If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the labeling time or decreasing the labeling temperature. If you label at 4°C we recommend overnight incubation.

Troubleshooting for Cellular Labeling 

No Labeling
If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is confirmed, check for expression of the CLIP-tag fusion protein via Western Blot using Anti-SNAP-tag Antibody (NEB #P9310). This antibody shows high crossreactivity with the CLIP-tag and can be used for Western Blot detection. Alternatively, CLIP-Vista Green (NEB #S9235) can be used to confirm presence of CLIP-tag fusion in cell extracts following SDS-PAGE, without the need for Western blotting.

Weak Labeling
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CLIP-tag substrate and/or the incubation time. Improving the protein expression may also improve the signal. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling.

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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