The Protein Man's Blog | A Discussion of Protein Research

Carbohydrate affinity chromatography is a method of choice for purification of glycoproteins, lectins and other carbohydrate metabolite proteins. This affinity chromatography uses carbohydrate ligands such as carbohydrates, glycoproteins and carbohydrate matrices for purification. The matrix needs to be activated for covalent immobilization of the ligand. Various methods used for the coupling of ligands depending upon the activating reagents are as follows:

---

 

INTRODUCTION:

Isolation of glycoproteins from protein solutions is routinely performed on concanavalin A (Con A) agarose (or sepharose). Con A is used for the purification of glycoproteins, polysaccharides and glycolipids as it binds molecules containing α-D-mannopyranosyl, α-D-glucopyranosyl and sterically related residues. Con A agarose has also be used in other application areas including purification of enzyme-antibody conjugates, purification of IgM and separation of membrane vesicles.

Con A is a metalloprotein and to maintain its binding characteristics the presence of both Mn²⁺ and Ca²⁺ is essential. Each subunit of Con A utilizes one calcium and one manganese ion and these cations can be removed under acidic conditions abolishing the carbohydrate-binding activity.

For the elution of bound molecules the preferred method is to use competitive eluents.  Suitable eluents include, but a re not limited to:

• methyl-α-D-mannopyranoside [50-200mM]
• methyl-α-D-glucopyranoside [50-200mM]

Common Elution Techniques are Ineffective!

 Researchers routinely report that they have issues with eluting their protein as seen by lower than expected yields.  This reduced yield also reduces the column capacity.

Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state.  HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.

It is interesting to note that while detergent-solubilized membrane proteins can be purified and concentrated through phase separation (which is a simple, efficient and cost-effective method that can be used for this purpose), most membrane protein scientists do not use this method or even know that such methods exist. To better understand and appreciate how this method works, here are some things you definitely need to know.

Phase Separation: Understanding the Basics

In a nutshell, phase separation (also known as cloud point extraction) refers to the state wherein the micelles in an aqueous solution become immiscible with water and form large aggregates that will separate from the water phase. This condition is usually achieved upon the addition of detergents or when the temperature or salt concentration of the solution is altered.