Protein Labelling
Small molecules such as fluorophores, biotin or click chemistry handles add new functions to proteins, allowing them to be tracked, quantified or immobilized.
Experimental data
a SDS gel of a biotinylation reaction. The tagged protein (lower band) is converted into the biotin-labelled protein (upper band).b Quantification of biotinylation over time.
How to use
Produce a Lock- or Key-tagged protein.
Mix it with the labelling reagent and the Connectase ligation reagent.
Incubate for 1 hour.
- Lock-Tag
- ELASKDPGAFDADPLVVEI
- C-terminal, 19 aa
- Key-Tag
- AGAFDADPLVVEI
- N-terminal, 13 aa
FAQ
How can I use Connectase to conjugate my protein?
You need to produce your protein recombinantly, with either a C-terminal Lock-Tag (ELASKDPGAFDADPLVVEI) or an N-terminal Key-Tag (AGAFDADPLVVEI). The recombinant protein can then be mixed with Connectase and a suitable conjugation partner.
Do I need a spacer between the tag and my protein?
For sterically demanding proteins, we recommend an extra arginine (R) N-terminal to the Lock-Tag and two extra alanines (AA) C-terminal to the Key-Tag. Many other conjugations work efficiently without any additional linker sequence.
Which buffers does Connectase work in?
Connectase works over a wide pH and salt range and tolerates common additives such as detergents and chaotropes. It has no special buffer requirements.
How long do I need to incubate?
Most reactions are finished after about one hour at room temperature. Higher temperatures speed up the reaction.
Do I need to remove the enzyme after the reaction?
In most cases, no. Connectase is used at low concentrations and catalyzes nothing beyond the conjugation. Where removal is needed, its His-tag allows capture on Ni-NTA resin.