Guidelines for Protein Purification

 Guidelines for Protein Purification


Protein characterization involves the use of experimental methods that allow for the detecting and isolation of a protein and its purification. The characterization of the structure and function of a protein or gene product is usually the next step. The success of the methods and techniques used often depends on the proficiency of the person performing the purification as well as the proper selection of the purification approach. 

These general guidelines listed here are applicable to any purification process.

Define goal or objectives   

Determine purity, activity and quantity required for the final product to avoid using the wrong method.  

Define properties of target protein and critical impurities 

Needed to simplify technique selection and optimization.

Develop analytical assays

Needed for fast detection of protein activity, recovery and critical contaminants.

Minimize sample handling at every stage

Important to avoid lengthy procedures which may risk losing activity or reduce recovery.

Minimize use of additives

Additives may need to be removed in an extra purification step or may interfere with activity assays.

Remove damaging contaminants early

For example, proteases that can break down the target protein may need to be removed or inactivated.

Use a different technique at each step

This takes advantage of sample characteristics that can be used for separation by combining methods that separate by size, charge, hydrophobicity,  or ligand specificity.

Minimize number of steps

Extra steps will reduce yield and increase time therefore combine steps logically.


=== Keep it simple!  ===


Apply the rule of three:


Minimize sample handling

Minimize use of additives

Remove damaging contaminants early


In addition, the requirement for how pure a protein needs to be is determined by the application it is indented to be used for.

Examples are shown below.





Extremely high

> 99

Therapeutic use, in vivo studies


95 to 99

X-ray crystallography and most physico-chemical characterization methods


< 95

Antigen for antibody production or N-terminal sequencing