Question:
What are some important applications of solubilized proteins?
The Protein Man Says:
In a nutshell, protein solubilization refers to the process of transforming non-soluble forms of proteins into a homogenous solution to facilitate successful electrophoretic separation. During the solubilization process, the chemical interactions that hold the aggregates together (including disulfide bonds, hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic interactions) are broken down to prevent protein aggregation or precipitation which may ultimately result in artifacts and/or sample loss.
The Importance of Solubilizing Proteins
In its native aggregated state, proteins tend to clog fine mesh filters. In addition, the particulate matter produced by protein aggregates does not pass through a gel permeation matrix and has an increased tendency of running in the void. These things can seriously affect the integrity of subsequent downstream applications so you really need to solubilize your protein samples using the most appropriate method.
Basically, your sample needs to be solubilized before the structure and the mechanism of action of your target protein can be studied and analyzed. However, since proteins vary greatly in size, charge, and water solubility, there is no single protocol that can be used to separate all types of proteins. As such, the specific solubilization method that should be used will then depend on the type of sample, the specific protein of interest, the amount to be separated and the subsequent use of the protein sample.
Protein solubilization is ideally carried out in the sample buffer that is compatible with your chosen electrophoresis technique. This is especially true when native electrophoresis is the method of choice. If this is not possible, proteins must be prepared in sample solubilization solutions which contain a number of compounds (this may include chaotropic agents, detergents, reducing agents, buffers, salts, and ampholytes) that are chemically and electrically compatible with the chosen electrophoretic technique. In such cases, you need to dilute your sample with concentrated electrophoresis sample buffer to yield a 1x final buffer concentration.
Applications of Solubilized Proteins
There are a number of downstream applications that necessitate the use of solubilized proteins. They are suitable for enzyme assays, electrophoresis, folding studies, chromatographic studies and many other downstream applications.
Electrophoresis
In preparing samples for SDS-PAGE or 2D electrophoresis, you need to remove the large particulate matter and dissolve your protein sample into a homogenous solution. Solubilizing your proteins will break all macromolecular interactions (non-covalent chemical interactions and disulfide bonds), facilitate easy removal of all substances that may interfere with the electrophoretic process (such as protein-RNA/DNA and protein-protein interactions and metabolites) and keep your proteins of interest in the solution as they approach their isoelectric points.
Remember, you should be able to extract all the proteins in your sample to obtain statistically significant quantitative data and correlate your results with experimental perturbations and resulting biological responses.
Chromatographic Studies
In most cases, recombinant proteins accumulate intracellularly in insoluble aggregates known as inclusion bodies. While there are cases where the expression of recombinant proteins in inclusion bodies can be advantageous, these proteins are mostly inactive and denatured, and may contain dimers and multimers. Since you need to recover high amounts of soluble and biologically active proteins to accomplish your task, you need to solubilize the proteins in these so-called inclusion bodies. If you do not need to fold your target protein, this can be done with urea but if your application calls for properly folded protein, you may need to use additives to break up the precipitate.
Enzymatic Assays
Since proteins work by interacting with other molecules, proper solubilization of the protein sample is required to determine specific activity and produce reproducible data. Solubilizing your protein sample will allow you to study the structure and the mechanism of action of your target protein. However, you may need to screen different solubilization reagents and solubilization techniques to transform the protein aggregate into the properly solubilized state.
