Some comments:

A positively or negatively charged protein is required to perform ion exchange chromatography, for adsorption to occur. Gel filtration or affinity chromatography (IMAC for example) do not require a particular protein charge for adsorption to occur. Hydrophobic interaction chromatography will be favored at the protein's pI, still you require a high salt concentration. A pH value equal to the protein's pI provokes a reduced protein solubility, still it will not precipitate if your protein concentration is below this value. Aggregation will always occur if proteins molecules violently collide with other protein molecules. Agglomeration occurs when these collisions are soft.

Buffers are comprised of several components - usually a buffering agent (such as Tris-HCl), NaCl, imidazole (in the case of IMAC), and sometimes glycerol, detergents such as Triton X-100, and a reducing agent such as DTT. Each component serves a different purpose. The NaCl is for preventing non-specific binding of proteins to the column while allowing the target protein to bind the column. Glycerol helps to prevent aggregation. Detergents help to solubilise proteins. Reducing agents prevent undesirable formation of disulphide bonds between cysteine residues which may cause aggregation.

Proteins are generally less soluble at their pI value, which is the pH at which the protein has no net charge. The general rule for keeping the protein stable is that the pH of the buffer should be within 1 pH unit of the pI. So you wouldn't want to risk having your buffer at pH 5.3.

Affinity chromatography and gel filtration do not require the protein to be negatively charged. In a purification procedure the choice of buffer pH is more about what keeps the protein soluble. Usually buffer pHs are based on physiological conditions so are in the pH 7-8 range.

For IMAC resin, a mildly basic pH (e.g. 7.5-8) is preferred. Acidic pH can cause the resin to lose the immobilized metal ions because metal chelation is accomplished by carboxylic acid groups.

For anion exchange chromatography, a pH above the pI is desired to impart a negative net charge. For cation exchange chromatography, a pH below the pI is desired to impart a positive charge. However, this is not an absolute rule. For one thing, you can use ion exchange chromatography as a subtractive step, whereby the protein of interest flows through but some of the contaminants bind. In such a method, the choice of pH may be different from when you want the protein to bind.

You also have to consider the stability of the protein, which can be affected by pH. Most proteins are most stable at near-neutral pH, unless they are normally found in an environment in which the pH is far from neutral.

Chromatofocusing is a chromatography technique that takes advantage of the different pIs of proteins by eluting with a pH gradient. pH gradient elution can also be used with ion exchange columns, although it is more common to use salt gradients.

In general, pH is not a factor for gel filtration chromatography, which separates on the basis of size and shape, unless the protein's oligomeric state or stability is affected by the pH.

It is usually considered undesirable to purify proteins at their pIs because of the risk that the protein will precipitate when its net charge is zero. However, this also is not an absolute rule. The key phenomenon associated with a protein at its pI is lack of mobility in an electric field, which is the principle of isoelectric focusing, rather than insolubility.

Dear Rosario

the definition of isoelettric point is:

the pH at which a particular molecule carries no net electrical charge. The net charge on the molecule is affected by the pH of its surrounding environment and can become more positive or negative due to the gain or loss of protons, respectively.

Since water is a polar solvent, the solubility of proteins in standard non-denaturing water buffers as PBS, Tris, hepes, MES, decrease when the pH become close to the pI.

In general we tend to work on a pH that is almost 1,5-2 unit different from the pI (you have to consider that pI value is theoretical since it is based on primary protein sequence, but in realty a folded protein expose in the surface only a fraction of its aminoacid and the real pI can be little different from the theoretical one)

In your case since you have a protein with low pI and work at pH>7 is preferable.

Manuele