Purification or removal of NAD+-dependent dehydrogenases and ATP-dependent kinases

Blue Sepharose 6 Fast Flow, Capto Blue, Capto Blue (high sub)

NAD+-dependent dehydrogenases and ATP-dependent kinases are members of a group of proteins that will interact with Cibacron Blue F3G-A, a synthetic polycyclic dye that shows certain structural similarities to the cofactor NAD+. When used as an affinity ligand, Cibacron Blue F3G-A will bind strongly and specifically to a wide range of proteins. Some proteins bind specifically due to their requirement for nucleotide cofactors, while others such as albumin, lipoproteins, blood coagulation factors, and interferon, bind in a less specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand. For details about Blue Sepharose 6 Fast Flow, Capto Blue, and Capto Blue (high sub), see Purification or removal of albumin in this chapter.

Performing a separation

Blue Sepharose 6 Fast Flow, Capto Blue, Capto Blue (high sub)

The information supplied in Purification or removal of albumin earlier in this chapter is applicable also to the purification of enzymes with an affinity for NAD+, but note the following:

For elution, use low concentrations of the free cofactor, NAD+ or NADP+ (1 to 20 mM), or increase ionic strength (up to 2 M NaCl or KCl, 1 M is usually sufficient).

For less specifically bound proteins: use higher concentrations of cofactor or salt or more severe eluents such as urea or potassium isothiocyanate. Polarity reducing agents such as dioxane (up to 10%) or ethylene glycol (up to 50%) may be used.

Materials