Must maintain biological conditions of pH, salt concentration and temperature to keep protein form denaturing and losing its biological activity. Surface Charge Properties can be used in ion exchange chromatography , where the charge on the protein depends on its pI and the pH of buffer used. Gel filtration takes advantage of protein size and shape to separate it from other proteins. The gel is a molecular sieve where large proteins exit from the column first, followed by medium size proteins and finally small proteins elute. If the gel filtration column is calibrated with proteins of known native molecular weight, then one can estimate the protein s native molecular weight by comparison to the standard proteins using a calibration curve.
Affinity chromatography takes advantage of the biological activity of proteins for separation.
Next elute the enzyme by adding a buffer to the column containing the substrate the free substrate in solution binds more tightly to the enzyme than the substrate covalently linked to the gel. The enzyme-substrate complex can be dialyzed to remove the substrate or the complex can be passed over a gel filtration column where the substrate will elute after the enzyme. This method separates proteins based on charge density while maintaining biological activity. The proteins separated on the Native PAGE gel will retain their biological activity or enzyme activity and this can be detected by using an appropriate activity stain. If you find you have only one protein on the gel and it stains for both enzyme activity and protein, then you have homogeneous protein and do not need to do more purification.