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 vials. Oligonucleotides for protein labeling using the enzymatic reaction of Sfp synthetase. Store all materials at -80°C.

Biotinylated and digoxigenin-labeled DNA handles (529 bp):

4 μl, 10 vials. Handles mix (50/50) with an overhang complementing the CoA-modified oligonucleotides. Store all materials at -80°C.

Sfp enzyme:

5 μl, 2 vials. Store all materials at -80°C.

10x Sfp reaction buffer:

50 μl | 10x. Store all materials at -80°C.

TCEP (100 mM):

5 μl, 2 vials | 100 mM. Reducing agent. Store all materials at -80°C.

6xHis AdK control protein (cysteine):

15 μl | 1.25 μg/μl (50 μM). Adenylate kinase protein with two fused ybbR peptides that can be used as a quality control.

Streptavidin-coated silica beads (⌀ 1.0–1.4 μm):

25 μl | 1% (w/v). Beads in PBS with 3 mM sodium azide, with a specific diameter (e.g., 1.2 μm) within the given range. Store all materials at +4°C.

Anti-digoxigenin-coated polystyrene beads (⌀0.7–0.9μm):

60μl| 0.1% (w/v) . Beads in PBS with 3 mM sodium azide, with a specific diameter (e.g., 0.8 μm) within the given range. Store all materials at +4°C.

Download the Full Specifications or View the Experimental Protocols

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.

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