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

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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.

While both monoclonal and polyclonal antibodies can be used in a wide variety of applications including Western blot, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, immunofluorescence, immunocytochemistry, Biochip technology and in the diagnosis of disease, they each have their own advantages which make them useful for different applications. To determine which type of antibodies should be used for a particular application, let us try to understand the difference between the two.

Recombinant antibodies (rAbs) are monoclonal antibodies which are generated in vitro using synthetic genes. Unlike monoclonal antibodies (mAbs) which are produced using traditional hybridoma-based technologies, rAbs do not need hybridomas and animals in the production process.

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INTRODUCTION:

Many protein modification reagents for biotinylation and cross-linking involve reactive groups that conjugate through primary amines.  Primary amines are found in all proteins and peptides as they make up the N-terminus and are also a component of the lysine residue side chains.  Amines, lysine ε-amines and N-terminal α-amines, are the most abundant group in protein molecules and represent the most common target for biotinylation. For example, BSA contains 59 primary amines, of which up to 35 are available on the surface of the molecules and can be reacted with amine reactive esters.

The most widely used amine reactive biotinylation and cross-linking reagents are the water insoluble N-hydroxysuccinimide (NHS) esters or the water soluble N-hydroxysulfosuccinimide (sulfo-NHS) esters.

The addition of a charged sulfonate (SO^3-) on the N-hydroxysuccinimide ring of the sulfo-NHS esters results in their solubility in water (~10mM), but are not permeable to plasma membranes. The solubility and impermeability to plasma membranes makes them ideal for studying cell surface proteins as they will only react with the protein molecules on the outer surface of plasma membranes.

Both forms of the NHS esters hydrolyze rapidly in aqueous solutions (7 hours at pH7, minutes at pH9) and for this reason the NHS esters must be handled and stored appropriately.  The NHS esters should be stored desiccated and by allowed to warm to ambient temperature before opening to avoid condensation.  That being said, repeated opening and closing and inappropriate handling will lead to the introduction of moisture and hydrolysis of the NHS-esters.

Hydrolysis (and conjugation) results in the release of NHS that can be assayed with the following procedure.