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

The serine protease trypsin (Tn) is commonly used in most proteomics experiments to digest proteins into peptides which can be analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).

When you lyse cells, either through physical disruption or by adding detergents, you also release a variety of other proteins (proteases) that are potentially harmful to your target protein. These proteases can cleave the peptide bonds between the amino acids of polypeptide chains and leave you with multiple short peptide fragments. When this happens, you will end up getting erroneous results from your experiments (western blotting, reporter gene analysis, protein interaction or activity assays).

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INTRODUCTION:

Knowing the amount of dye conjugated to a protein is essential for predicting the amount of dye required for an experiment and for ensuring good control of fluorescence between experiments.

The following blog post explains how to calculate the degree of protein labeling by a selection of dyes by using protein and dye absorbances.

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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.