EK-Away™ Resin, 30 mL - FAQs

View additional product information for EK-Away™ Resin - FAQs (R18002)

4 product FAQs found

Is the EK-AWAY resin reusable?

Yes, the EK-AWAY resin is reusable. Wash resin twice with 2 times the bed volume of stripping buffer (100 mM formic acid, pH 3.0, and 100 mM NaCl) to elute off any bound peptides. For each wash, rock sample for 2 minutes. Pellet resin by centrifugation for 1 min at 800 x g and discard the supernatant.
To re-equilibrate the resin, wash resin twice with 2 times the bed volume of binding buffer (50 mM potassium phosphate, pH 8.0, and 500 mM NaCl) by rocking for 2 min. Pellet resin by centrifugation for 1 min at 800 x g and discard the supernatant. The resin in now ready for rebinding.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Will DTT, Triton X-100 detergent, Tween 20 detergent, Thesit, calcium chloride, sodium chloride, or SDS affect the efficiency of Enterokinase (EKMax) enzyme cleavage?

Enterokinase is active in buffers containing up to 1 mM DTT, 0.1% Triton X-100 detergent, 0.1% Tween 20 detergent, and 0.1% Thesit. It is recommended to have 10mM Tris pH 8.0 and 10 mM calcium chloride in the buffer. Enterokinase is inhibited by sodium chloride and SDS.

Should Enterokinase be resuspended in a buffer containing 50% glycerol to protect the protein from freeze/thaw cycles?

Freeze thaw has a minimal effect on the activity of Enterokinase. The addition of glycerol is not necessary but can make handling of the enzyme easier.

How specific is cleavage by EKMax Enterokinase? Are there any alternate cleavage sites for the enzyme?

Enterokinase cleaves after the sequence (Asp)4-Lys.

It has been proposed that the active center of enterokinase possesses a distinctive cationic subsite that binds -(Asp)4. Enterokinase is highly specific and tolerates very few changes to its recognition site. If the ionic charge of the recognition site is preserved, enterokinase will recognize the site, but the rate of hydrolysis of the peptide bond will be reduced (Light and Janska, 1989). The four aspartyl residues act as a signal for enterokinase cleavage. It has been reported that with only three aspartyl residues the rate of hydrolysis is reduced. Two aspartyl residues preceding the lysyl residue are the minimum number of acidic residues needed to maintain specificity (Maroux et al., 1971). Non-specific cleavage by enterokinase may occur in the cases described above, but this is usually alleviated by reducing the amount of enzyme used.