Frequently asked questions
Using Connectase
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.
Protein Detection
How can I quantify my protein?
We supply a defined protein standard that you can add to your sample. The signal ratio between the standard and your protein then gives you the protein concentration.
Which fluorophores can be used for the Protein Detection assay?
We currently supply Cy5.5 (Ex/Em ≈ 675/694 nm) and Cy5 (Ex/Em ≈ 649/667 nm) and plan to expand the set of fluorophores in the future.