Proteomics/Protein Sample Preparation/Contaminant Elimination< Proteomics | Protein Sample Preparation
In almost any biological experiment, eliminating contamination is of utmost importance. This is especially true when dealing with proteins, as human subjects can contaminate the sample with their own proteins, leading to unexpected results. Dialysis, Desalting, Protein Precipitation and Ultrafiltration are all methods that may be used to prevent unwanted molecules, assuring accurate and reliable results.
Dialysis is a common protocol for isolating proteins in a sample based on size exclusion. Semi-permeable membranes are manufactured with varying pore sizes such that a dialysis bag can be selected specific to a particular experiment’s needs. The protein sample, in solution, is sealed in the bag which is then placed in a large volume of buffer, normally 200 – 500 times the volume of the sample . Due to the gradient between the sample and the buffer, the molecules in the sample attempt to flow from the dialysis bag where only those smaller than the pore size actually leave the sample . The power of dialysis lies in its ability to cleanse a sample of a number of contaminants at once, including small proteins, and small contaminating molecules such as salt.
The key to a successful separation based on dialysis is the selection of an optimal pore size. Membrane pore sizes are typically measured in Daltons such that pores measuring 1,000 Daltons will retain molecules a molecular mass of 1,000 Daltons. More information on preparing a dialysis tube based separation can be found here.
Another method for removing foreign molecules from protein samples, desalting provides us with the ability to remove detergents, DNA or RNA, lipids and salts. Oftentimes, this method is known as gel filtration, or molecular exclusion. The premise behind desalting is that the molecules in the sample are passed through a porous resin. The smaller molecules pass into the pores and get stuck, whereas the larger molecues do not fit and simply flow around and out of the resin. In doing so, the larger molecules are separated away from the other molecules in the solution, which remain stuck in the pores.
Similar to the other methods already discussed, precipitation involves removing unwanted substances from our protein sample prior to analysis. In this case, it is the protein that is precipitated, allowing for the remaining solution to be poured off. Most often, precipitation is achieved using salts.
At low salt concentrations, proteins are generally observed to be more soluble than at high concentrations. Using this information, a technique known as salting out is often used which involves increasing the salt concentration until the proteins precipitate out. Although this is the most common form of protein precipitation, similar results can be obtained by changing the pH of the solution or through the introduction of metal ions.
Ultrafiltration is a process similar to gel filtration or molecular exclusion, but also relies on pressure in separating the molecules. Due to the pressurized samples, a much smaller pore size may be used (around 250 nanometers). The smaller molecules are then forced through the pores while the larger proteins are retained.