Collagen Ligands Collection
The successes of the Collagen Ligands Collection
To date 170 sites where over 30 proteins bind to collagen II have been mapped,
providing firm conclusions about the amino acid distribution within such binding sites.
Many CLC peptides bind more than one protein. For example, ligand CLC-II-44 is highly promiscuous; it alone binding over 20 different proteins.
The Ligands have been used to determine atomic level resolution of interactions between collagen and other proteins, advancing our understanding of ECM assembly for applications such as tissue engineering.
Collagen Ligands Collection Design
The collagens are fundamental components of the extracellular matrix, and are highly interactive with many partners within the ECM and several sets of receptors on the cell surface.
Collagen-like peptides open the way to rapid and
independent verification of putative protein-collagen binding sites.
The scientists at Triple Helical Peptides Ltd developed the Collagen Ligands Collection (previously described as collagen Toolkits) within the University of Cambridge
and have synthesised these peptides for over 20 years.
The Ligands encompass the entire triple-helical domains of collagens II and III can be used to
map the binding of receptors and other proteins onto the tropocollagen molecule
(the triple-helical collagen monomer).
The Guest sequences of the Ligands are each 27 residues long; initiating at the N-terminus of the collagen COL domain and advancing successively by 18 residues,
allowing a 9-residue overlap between adjacent peptides. The collagen primary sequence is flanked on each side by five GPP triplets (the Hosts)
to ensure triple-helical conformation. GPC extensions on each end of the Ligand allow crosslinking of peptides (if desired),
making each 63 residues long.
Collagen Ligands Collection III comprises 57 peptides, and II, 56 peptides.
The triple-helical but inert ligand GPP10 is also included as a negative control.
GPC-GPPGPPGPPGPPGPPGPPGPPGPPGPPGPP-GPCG-NH2
At present, we are able to supply Collagen II and Collagen III as sets of 56 and 57 peptides,
in triplicate 96-well plates each arranged as in the schematic below (It is an example. The plates you get always have their unique description and scheme.).
Plates can be shipped to end-users for use in their own labs to map binding sites within these collagens.
We add three additional plates for optimisation process.
The Collagen Ligands Collection comes pre-coated on 96 well plates, in a ready-to-use adhesion assay format,
and can be applied in solid-phase binding assays to map sites where collagen receptors and extracellular
matrix components bind to collagens. Once a binding site is located,
cycles of truncation and substitution allow exact residues involved to be determined,
and corresponding minimal peptides to be synthesised for use
in structural and functional studies.
Simple Assay Protocol
Collagen Ligands are pre-coated on the plate at a saturating concentration of 10 µg/ml.
1. Block all wells to be used with 200µL 50 mg/ml BSA in Tris-buffered saline (TBS) for 1h at room temperature.
2. Wash all wells three times with 200µL adhesion buffer (1 mg/ml BSA in TBS containing 0.1% (v/v) Tween-20).
3. To each well, add 50-100µL target protein. We recommend a starting concentration of 100nM, but this may require optimisation. Leave for 1h at room temperature.
4. Wash all wells three times with 200µL adhesion buffer.
5. To each well, add 100uL primary antibody against your target protein in TBS containing 0.1% (v/v) Tween-20. We recommend an antibody dilution factor of 1:2000, but this may require optimisation. Leave for 1h at room temperature.
6. Wash all wells three times with 200µL TBS containing 0.1% (v/v) Tween-20.
7. To each well, add 100µL enzyme-conjugated secondary antibody (of your choice) in TBS containing 0.1% (v/v) Tween-20. We recommend an antibody dilution factor of 1:10,000, but this may require optimisation. Leave for 1h at room temperature.
8. Wash all wells three times with 200µL TBS containing 0.1% (v/v) Tween-20.
9. Add substrate as appropriate and according to the manufacturer's instructions.
FAQs
How should I store the plates?
The plates should be stored with plate sealers intact at 4°C.
How long can I store the plates?
The plates are shipped at ambient temperature and can be stored at 4°C for up to three months.
Can I store the plates in blocking buffer?
No. To avoid microbial growth, we recommend storing the plates 'dry' as received until used.
What blocking agent should I use?
We recommend 50mg/mL BSA in TBS.
What is TBS?
We recommend 50 mM Tris-Cl, 150 mM NaCl, pH 7.4.
What concentration of target protein should I use?
We recommend 100nM, but optimisation may be required.
Can I use different substrate conjugated antibodies?
Yes, the substrate can be tailored to your secondary antibody conjugate.
Can I re-use the plates?
No. The plates are single use only and cannot be stripped or re-used.
Can I get xCELLigence plates coated with peptides from Collagen Ligands Collections for studies with cells or bacteria?
YES. We can coat AGILENT's xCELLigence plates (E-PLATE 96 PET).
Troubleshooting
Problem: Weak/no signal.
Solution:
1. Omission of key reagent.
2. Washes too long/stringent.
3. Increase incubation time of target protein or antibody.
Problem: High background
Solution:
1. Decrease target protein concentration.
2. Decrease antibody concentration(s.
3. Optimise blocking buffer.
4. Increase percentage of Tween-20 or use alternative detergent.
5. Increase wash times between steps
Problem: Uneven colour development
Solution:
1. Incomplete/uneven washing of wells.
2. Pipetting (user) error.
Download the CLSs' white paper to get these deatils