Protein tethering kit – Cys

$818

Prefer to order through purchase order? Request a quote at the checkout page.

 

This kit uses cysteine-maleimide chemistry to attach DNA handles to proteins of interest and perform optical tweezers measurements of protein folding properties and protein conformational changes.

In a first step, short maleimide-modified oligonucleotides (36 bp) react with specifically incorporated cysteines of the protein. In a second step, the protein-attached oligonucleotides anneal with complementary 5’-overhangs of two 520 bp DNA handles, to form a protein–handle complex that is ready for tethering. The kit includes purified AdK with specifically incorporated cysteines that can be used as positive control and quality assurance for each step of the labeling 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:

  • Maleimide-modified oligos (36 bp)
  • Biotin- and digoxigenin-modified DNA handles (520 bp per handle)
  • Reducing agent TCEP
  • 6xHis tagged AdK protein
  • Buffer 40 mM Tris HCl, 50 mM NaCl
  • 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

Maleimide-modified oligonucleotides

Lyophilized (Dried transparent pellet, store at -20), 2 tubes for the labeling of 2 proteins – each tube contains 9 nmol of maleimide-modified oligonucleotide. Store at -20°C. Upon labeling, protein–oligonucleotide 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. Aliquot, snap freeze, and store at -80°C.

TCEP

40 μl – 10 mM reducing-agent TCEP sufficient for labeling 20 proteins with maleimide-modified oligonucleotides. Aliquot and store at -20°C.

6xHis AdK protein

35 μg – 6xHis tagged AdK protein with a cysteine incorporated at positions 42 and 144 of the protein as previously described (Pelz et al (2016) Nature Communications). This amount of protein is sufficient for 2 labeling attachment experiments. Store at -80°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) anti-digoxigenin coated polystyrene microspheres with a diameter of 0.75 µm. Binds  digoxigenin-modified handles. Store at 4°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.