This method involves filtering a diluted lysate or protein solution through a semi-permeable membrane, so that proteins larger than the diameter of pores punctuating the surface of the membrane retained and only the small ones are extruded. Dialysis can also be used to concentrate the protein solution; however, because this method cannot efficiently differentiate proteins, it is often followed by other more specific techniques. Automated systems now exist that can carry out dialysis, and several commercial kits also provide the necessary reagents (solutions).
Proteins do not dissolve (or "solubilize") well in solutions of high salt concentrations. This property of solubility will differ between proteins and is a good way to distinguish between closely related ones. From a solution of several proteins, increasing amounts of salts like ammonium sulfate can be used to fractionate and precipitate the larger proteins first (at lower ammonium sulfate levels), and therefore also to concentrate very dilute samples. This is the most commonly used method due to its simplicity.
This includes gel-filtration, ion-exchange, affinity and high-pressure liquid chromatography (HPLC). In all cases, the lysate is allowed to flow through a column containing porous beads of different, but specific characteristics. Gel-filtration chromatography to separate small proteins from larger ones, uses beads made from materials such as dextran, polyacrylamide or agarose. Ion-exchange chromatography separates proteins by net charge using beads that have carboxylate groups on them. Proteins that stick to the beads are retained, while those that bind weakly or not at all flow straight through and are collected out the other end of the column. It is also possible to recover the protein that was bound. This approach is used in affinity chromatography, which exploits a protein's affinity for specific chemical ligands. The bound protein is washed off from the column by flooding the beads with a solution to decrease the binding. HPLC is a higher resolution, faster and much-improved version of chromatography.
Solutions of proteins of various masses or densities may be separated based on the time it takes to sink or pellet to the bottom of a tube during centrifugation. Heavier and/or denser particles will pellet first, while lighter or less dense ones will remain dissolved. Separation of proteins is carried out in a solution containing a layers of increasing or decreasing concentration of material, such as sucrose or some other media, like Percoll. Ultracentrifugation in this "concentration gradient" allows separation of large proteins from smaller ones. Both the pellet and the supernatant (containing the smaller proteins) can be collected for further purification or analysis.
Good isolation methods will yield large quantities and high purification proteins, and in each case, subsequent experimental requirements should be taken into account when selecting the most appropriate method to use. Good resources for more detail and precise protocols are Clive Dennison's 'A Guide To Protein Isolation' (Kluwer, 2003) and Hafiz Ahmed's 'Principles And Reactions Of Protein Extraction, Purification, And Characterization (CRC Press, 2005). However most protocols and professional laboratories use commercially developed kits that consist of simple and efficient technical steps, but more importantly, high purity, for isolation of various classes of proteins.