Tris NTA (Tris Nitrilotriacetate) Oligo Conjugate

Gene Link provides custom synthesis of NTA Tris (Nitrilotriacetate) Oligo Conjugate.
NTA-Tris modification is a post synthesis conjugation to an active NHS group of either a NHS-Carboxy C10 or a NHS-dT group thus an additional modification is required at the 5' end with additional charges for that modification.

Yield of Post Synthesis NHS, Maleimide & Click Ligand Conjugation*

Oligo Scale of Synthesis	Yield, nmols
50 nmol	2 nmol
200 nmol	5 nmol
1 umol	16 nmol
2 umol	30 nmol
5 umol	75 nmol
10 umol	150 nmol
15 umol	225 nmol
* The yield will be lower for oligos longer than 50mer. Click here for yield table of long oligos.
* Click here for RNA Oligos scale of synthesis and yield.
NHS Ligand conjugation requires a primary amino group. Gene Link offers a wide selection of amino modifications for 5', 3' and internal sites. Click here for a list of conjugation chemistry modifications.
Maleimide Ligand conjugation requires a thiol group. Gene Link offers a wide selection of thiol modifications for 5', 3' and internal sites. Click here for a list of conjugation chemistry modifications.
Click Chemistry Ligand conjugation requires a corresponding Click modification; examples Alkyne:Azide, Azide:DBCO, BCN:Azide, BCN:Tetrazine and TCO:Tetrazine. Gene Link offers a wide selection of click modifications for 5', 3' and internal sites. Click here for a list of click chemistry modifications.

Nitrilotriacetic acid (NTA) is widely used in affinity chromatography to purify His-tagged proteins. The NTA chelator forms a complex with metal ions, usually Ni(2+), which then binds to histidine residues in the His-tag. Two forms of NTA are available, monovalent (NTA Mono) and trivalent (NTA Tris) differ in their binding strength and application. Tris-NTA has a significantly higher affinity for His-tags compared to mono-NTA, with affinities in the nanomolar range versus the micromolar range for mono-NTA. This higher affinity is due to the multivalency of tris-NTA, which allows it to bind the His-tag at three points, resulting in a much more stable and strong interaction. This leads to more stable baselines in experiments like SPR and improved detection sensitivity.

Comparison of NTA Mono and NTA Tris Affinity & Other Features Towards His Tag

Feature	Mono NTA	Tris NTA
Structure	NTA Mono: One NTA group per ligand	NTA Tris: Three NTA groups per ligand (clustered)
Histidine Binding	Binds via 2-4 histidine residues	Binds via 6+ residues, higher avidity
Affinity	uM range K_D	nM-pM range K_D
Elution	Easy with imidazole/EDTA	Difficult due to strong binding. Requires 100 mM imidazole or higher
Application	Protein purification. Weak binding acceptable	Requiring high affinity binding. Biosensing, stable immobilization
The enhanced binding of tris-NTA is a result of multivalency, where the simultaneous binding of three NTA-metal ion complexes to the histidine tag creates a much stronger overall interaction.

Applications - NTA Mono is commonly used in immobilized metal affinity chromatography (IMAC) for routine purification of His-tagged proteins. - NTA Tris provides higher binding strength and is preferred in biosensor applications such as SPR (Surface Plasmon Resonance) and BLI (Bio-Layer Interferometry).

Binding Affinity Summary

Ligand	Approximate K_D to His tag
NTA Mono-Ni(2+)	~1-10 uM
NTA Tris-Ni(2+)	~1-100 nM
The enhanced binding of tris-NTA is a result of multivalency, where the simultaneous binding of three NTA-metal ion complexes to the histidine tag creates a much stronger overall interaction. The His-tagged protein is effectively "gripped" by three separate NTA-metal interactions. Dissociation requires breaking all three bonds simultaneously, which is statistically far less likely and requires much more energy.

References

1. 1. J. Shimada, T. Maruyama, T. Hosogi, J. Tominaga, N. Kamiya, M. Goto,. Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system, Biotechnol. Lett. 30 (2008) 2001-2006.
2.J. Shimada et al DNA enzyme conjugate that can detect thrombin. Anal. Biochem. 414 (2011) 103-108. - NTA Mono (Nitrilotriacetate) Oligo Conjugate
3. Hochuli, E., et al. (1988). Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. *Bio/Technology*, 6, 1321-1325
4. Lata, S., et al. (2005). High-affinity adaptors for switchable recognition of histidine-tagged proteins. *Journal of the American Chemical Society*, 127(29), 10205-10215.
5. Porath, J., et al. (1975). Metal chelate affinity chromatography, a new approach to protein fractionation. *Nature*, 258, 598-599.

- Tris NTA (Tris Nitrilotriacetate) Oligo Conjugate