Protein tethering kit (ybbR)

$818

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This kit applies a ybbR tag and Sfp enzyme to functionalize your protein of interest with DNA handles for subsequent optical-tweezers measurements of protein folding and conformational dynamics. The system constitutes an alternative to the Protein tethering kit (cysteine). The Protein tethering kit (ybbR) is the ideal option for applications where cysteine incorporation is not possible, for example, due to the presence of native cysteines on your protein of interest.

The system is based on the enzymatic reaction of Sfp synthetase that attaches CoA-modified oligonucleotides on specific serine positions on the ybbR peptide. Sfp catalyzes the covalent binding of a CoA-modified oligonucleotide to each of the two ybbR peptides inserted in the protein of interest. These oligonucleotides then anneal with the complementary 5’-overhangs of two DNA handles.

The kit also includes purified and functionalized adenosine kinase (AdK) that can be used as a positive control and quality assurance for each step of the labeling procedure.

Our Protein tethering kit relieves you from costs and time that are typically required for personnel training, troubleshooting, experiment optimization, and custom application development. After finishing this kit, new materials can easily be ordered as a recurring package.

Material supplied

  • CoA-modified oligonucleotides
  • Biotin-modified and Digoxigenin-modified DNA handles (520 bp each)
  • Sfp enzyme
  • 10 x Sfp reaction buffer
  • Reducing agent TCEP
  • 6xHis ybbR tagged AdK protein
  • Streptavidin-coated polystyrene beads (1.76 µm)
  • Anti-digoxigenin coated polystyrene beads (0.75 µm)
  • Estimated for 2 weeks of measurements
  • Estimated 1-3 weeks delivery time

Specifications

CoA-modified oligonucleotides

20 µl (2 aliquots) – CoA-modified oligos. Each aliquot contains the amount required for 1 protein labeling reaction. Store at -80°C. Upon labeling with oligos, protein–oligo chimeras can be snap frozen and stored at -80°C.

Biotin-modified and digoxigenin- modified handles

One tube containing an amount of handles mix sufficient for 10 labeling reactions. Upon arrival: aliquot, snap freeze, and store at -80°C.

Sfp enzyme

5 µl (2x, aliquots) – Sfp enzyme. Each aliquot contains the amount required for 1 protein labeling reaction. Store at -80°C.

Sfp reaction buffer

200 μl (10x) – Sfp reaction buffer.

TCEP

100 µl – 10 mM reducing-agent TCEP. Upon arrival: aliquot and store at -20°C.

Polystyrene streptavidin beads (1.76 µm)

25 µl – 1% (w/v) (typical dilution factor 1:1000) streptavidin coated polystyrene microspheres with a diameter of 1.76 µm. Binds to biotin-modified DNA handles. Store at 4°C.

Polystyrene anti-digoxigenin beads (0.75 µm)

250 µl – 0.1% (w/v) (typical dilution factor 1:100) streptavidin coated polystyrene microspheres with a diameter of 0.75 µm. Binds to digoxigenin-modified handles. Store at 4°C.

6xHis AdK protein

35 µg – 6xHis tagged AdK protein with ybbR fused to the N- and C- terminus. The protein amount is sufficient for 1 labeling experiment with DNA handles. Store at -80°C.

Product Resources

Want to learn more?

See our Protein Folding and Conformational Changes application notes.

Want to use this product in practice?

Find out more about the C-Trap here.