FAQs on Analysis with Affinity Chromatography

What is the support material for Tosoh Bioscience affinity resins?

TSKgel affinity chromatography columns are based upon the G5000PW methacrylate porous resin. This “5PW” media is hydrophilic, with 100 nanometer (nm) pores and an estimated protein size exclusion limit of 1000 kilodaltons.

What are the ligand concentrations, adsorption capacities and test analytes for the various affinity column supplied by Tosoh Bioscience?

Table 1: Characteristics of TSKgel affinity columns

Column packing	Ligand type	Ligand concentration	Adsorption capacity	Sample
Boronate-5PW	m-aminophenyl boronate	not available	40 µmol/mL resin	sorbitol
Chelate-5PW	iminodiacetic acid	20 µmol/mL resin	not available	not available
Tresyl-5PW	tresyl	ca. 20 µmol/mL resin	60 µmg/g dry resin (coupling capacity)	soybean trypsin inhibitor

Can I get a TSKgel Affinity LC column with non-metal wetted parts?

Yes. We supply glass columns in two formats: 5 mm ID x 5 cm L and 8 mm ID x 7.5 cm L with 10 µm pore size Pyrex frits.

The shipping solvents for these columns are as follows:

Boronate-5PW:	distilled water
Chelate-5PW:	acetate buffer
Tresyl-5PW:	acetone

Which affinity column should I use?

Table 2: Applications of TSKgel affinity columns

Group specific ligands	Application
TSKgel Boronate-5PW	carbohydrates, nucleic acids, nucleosides, catecholamines
TSKgel Chelate-5PW	immunoglobulins, transferrin, lectins, milk proteins, membrane proteins, peptides

Chemically activated ligands	Application
TSKgel Tresyl-5PW	coupling of ligands to form a custom affinity resin

How do I load and desorb protein from a TSKgel affinity column?

TSKgel Chelate-5PW is based on a 100 nm (1000 Å) pore size polymethacrylate base material covalentely bonded with iminodiacetic acid (IDA) groups. Prior to chromatography, a metal ion, such as Zn²⁺, Ni²⁺ or Cu²⁺, is added to form a chelate with the IDA group. Peptides and proteins containing histidine residues will normally adsorb to these chelated ions at neutral pH. The retained compounds can be eluted with buffer containing imidazole or glycine.
TSKgel Boronate-5PW is based on a 100 nm (1000 Å) pore size polymethacrylate base material bonded with m-aminophenyl boronate. This ligand is capable of forming a tetrahedral boronate anion under alkaline pH conditions. This anionic structure can bind with 1,2 cis-diol groups such as those found in carbohydrates, glycosolyated proteins, and catecholamines. Interaction between the boronate anion and the 1,2 cis-diol groups is enhanced in the presence of Mg2+ ions and is inhibited by amine-containing buffers. Adsorption onto the TSKgel Boronate-5PW matrix takes place in basic buffers such as HEPES and morpholine while desorption takes place in carbohydrate or amine-containing mobile phase like sorbitol or Tris.
TSKgel Tresyl-5PW is based on a 100 nm (1000 Å) pore size polymethacrylate base material derivatized with a 2,2,2-trifluroethanesulfonyl ligand. Unlike other TSKgel affinity columns, the TSKgel Tresyl-5PW columns require activation with a user-selected ligand containing amino, thiol, phenol, or imidazole groups. The resulting structure is literally a custom affinity ligand with excellent pH stability and minimal ligand loss due to leaching. Binding and elution conditions depend on the custom-specific ligand.