HOW TO CARRY OUT MEMBRANE PROTEIN ISOLATION
The topic of protein isolation is one that has already generated several authoritative articles, reviews, and books. Even though there are numerous methods that are applicable to the isolation and purification of proteins of all organisms, you are better off developing your own strategy for the isolation of the target protein.
Unlike DNA research, there are no standard protocols, or “recipes” or manual that you can follow that outlines a step-by-step approach that is applicable to the isolation of all proteins. Additionally, there are no procedures specific to any organism that will allow you to plan a course of action that’ll lead to a specific outcome. Protein isolation has been described as more of an art than a science.
Whatever method you come up with for the isolation of a given protein, make sure it aligns with the research objectives. For example, if you needed the isolated protein for analytical purposes, then you need a procedure that’ll give you modest amounts of relatively pure proteins. And if it is required for structural or physicochemical studies, then your procedure should be tailored to give you highly purified, homogenous products. The remarkable variation in the chemical and physical properties of a protein is what makes isolation – and purification – of a single protein from a cell that contains a mixture of thousands of unrelated proteins possible.
The characteristics unique to each protein include; solubility, net charge, size, amino acid composition, subunit structure, sequence, shape, hydrophobicity, heat-stability, ligand/metal binding properties, isoelectric point, and post-translational modifications. These characteristics are usually the ones exploited when formulating a method for the isolation of proteins. Some of the procedures formulated include:
- Chromatography – net charge, metal-binding sites, surface hydrophobicity
- Precipitation – solubility
- Centrifugation – shape, size
Generic outline for protein isolation
Generally, membrane protein isolation and purification essentially involves five types of steps
- Efficient extraction from biological material
- Separating the protein and non-protein components (lipids and nucleic acids)
- Precipitation steps – this is done initially to recover a bulk of the protein from the crude extract and then broken down into manageable factions
- Use of hydrophobic chromatography/size fractionation or ion-exchange chromatography for further extraction of the target protein from the bulk protein
- Several refined steps such as an “affinity” matrix to get maximum yields of a highly purified target protein.
To successfully evaluate the progress of the isolation and purification process, make sure to have a convenient assay procedure (based on some easily monitored property of the target protein or of enzymatic property) available at hand. Measuring the enzymatic activity using a colorimetric or spectrophotometric method is the most convenient since the progressive increase in specific activity is a great indicator of the effectiveness of the isolation and purification process. If the protein lacks any biological activity that can be easily measured, then your best bet is to use an immunochemical method such as ELISA (Enzyme-Linked-Immunosorbent Assay) or western blotting. This is only possible if there are suitable antibodies are present. If that is the case, then at each stage of purification, you’ll need to carry out an electrophoretic resolution of the protein population in samples.