Protein tethering kit – ybbR

$818

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This kit uses the ybbR tag and Sfp for protein functionalization with DNA handles. The system constitutes an alternative method to the cysteine–maleimide based DNA handle functionalization. You can use this option when the latter is not suitable due to the presence of native cysteines of the the protein of interest. The system is based on the enzymatic reaction of Sfp synthetase that attaches CoA-modified oligopeptides on specific serine positions on the ybbR peptide. Sfp catalyzes the covalent binding of a CoA-modified oligonucleotide (36 bp) 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.

Trapping is achieved by the simultaneous binding of the biotin-modified DNA handle to the streptavidin bead and the digoxigenin-modified DNA handle to the anti-digoxigenin bead.  The Kit includes purified AdK protein tagged with a ybbR, which you can use as positive control and quality assurance for each step of the procedure.

A schematic illustration representing three-domain protein tethered to two optically trapped beads through DNA handles. Extending or retracting protein structure through optical manipulation causes the protein to unfold and fold, respectively. Read more here: https://lumicks.com/application/protein-folding-optical-tweezers-fluorescence-microscopy/ 

Material supplied

  • CoA-modified oligonucleotides (36 bp)
  • 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 (handles and beads, more can be ordered as Protein handles kit (recurring))
  • Estimated for 8 weeks of measurements (labeled protein and control)
  • 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.

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