In proteomics, fractionation is a separation process in which a mixture of compounds is divided into smaller fractions according to a gradient. The gradient can be based on a specific property of a set of properties. Fractionation widely employed to separate substances of interest from other substances. Fractionation techniques can be applied at different levels.
An ideal homogenate contains a suspension of intact and individualized subcellular compartments. To collect individualized cells, we use chelating agents such as EDTA for enzymatic and mechanic disaggregation. Then we disrupt plasma membrane by detergents or mechanical methods such as ultrasonification.
While some claim cell fractionation and subcellular fractionation to be different, most researchers use the terms interchangeably. Cell fractionation refers to fractioning the different components of the cell. This is among the first steps in protein profiling.
Subcellular compartments can be separated based on their properties such as size, density, and charge. The first step is to separate the nuclei and the unbroken cells from cytoplasmic organelles by differential sedimentation at low centrifugal force in order to obtain postnuclear supernatant (PNS). The supernatant can then be separated using differential centrifugation based on size, weight, density or even shape. Differential centrifuagation is a time-dependent technique. Isopycnic centrifugation separates by density by passing the organelles through a sucrose gradient. Free-flow electrophoresis (FFE) separates by charge.[3] Immunoisolation techniques can be used to separate using the biological properties of teh organelles. Several commercial and non-commercial devices and techniques exist for this purpose.
The goal of sample fractionation is to improve the detection of low abundance proteins. The dynamic range for 2DE is limited to 104 while teh protein expression range is from 107 to 1012. Therefore, only the most abundant proteins are detected.
Increasing the amount of sample is not always a good solution. It is usually better to further subdivide the mixture. Fractionation methods are based on the physico-chemical properties of proteins. Following is a list some properties and techniques exploiting these properties.
| Property | Fractionation Method |
|---|---|
| Size/shape | size exclusion chromatography |
| Surface charge | ion-exchange chromatography |
| Isoelectric point | electrophoretic methods |
| Surface hydrophobicity | reverse phase chromatography |
| Binding specificity | affinity chromatography |
| Solubility | solvent extraction |
22 proteins comprise 99% of the protein mass in serum. Affinity chromatography can be used to remove albumin, immunoglobulins, etc. Antibodies and proteins A, G, and L are often used to remove these proteins.
Protein precipitation can be induced by using organic solvents such as acetone, salts such as ammonium sulfate, or by changing the pH. Protein precipitation is used for removal of large abundant proteins. This methods lack specificity.
Ultrafiltration is a pressure-driven, semi permeable membrane-based separation process. It achieves separation on the basis of size. The membrane retains larger molecules. This method lacks specificity.