In the previous post, we discussed about the entire procedure 2-D PAGE in brief. In this blog, let’s discuss the first step of 2-D PAGE, that is the Sample preparation, more in detail.
Sample Preparation in 2D-PAGE-
An appropriate sample preparation method is prerequisite to any technique, including 2-D PAGE.
The main purpose of sample preparation are complete solubilisation, disaggregation, denaturation and reduction of the proteins in the sample. The methods differ from sample to sample, it’s different for the soluble and the bound proteins and if the proteins are complexed with other protein or biomolecules. The procedure must be properly chosen and optimised for each sample type and the protein type.
The different steps included in the sample preparation are:
This is the first step when considering the analysis of intracellular proteins. Choice of the disruption method depends on if the sample is from cells, solid tissue, or other biological material. The method also depends on if the study is about all the cellular proteins or just a particular subcellular fraction.
Depending on the level of disruption the methods can be divided into Gentle lysis methods and Vigorous lysis methods.
– Gentle lysis methods don’t cause extensive disruption. Eg. Osmotic lysis, enzymatic treatment, freeze-thaw, detergent. It can also be employed when only one particular subcellular fraction is to be analysed.
– Vigorous lysis methods can cause greater disruption. Eg. Sonication, Ultrasonic waves, Grinding, Mechanical homogenization, Glass bead homogenization.
A special care should be taken of the protease in the cell, during the cell disruption. The proteases may cleave other proteins released. Hence the protein sample should be protected from proteolysis during cell disruption and subsequent preparation. This can be done by inhibiting the proteases by using strong denaturants such as 8 M urea, 10% TCA, or 2% SDS and low a temperature during the sample disruption.
In case, if only a subset of the proteins in a tissue or cell type are to be studied, prefractionation has to be carried out during sample preparation usually after gentle cell disruption. The subcellular compartment (e.g. nuclei, mitochondria, plasma membrane) can be purified by differential centrifugation or other means prior to solubilization of proteins for 2-DE.
Precipitation of the proteins in the sample help in the removal of interfering substances and concentration of the proteins as well. Precipitation of the proteins help removing the contaminating species such as salts, detergents, nucleic acids, lipids, etc. which can interfere with the 2-D result. For the concentration of the protein sample the precipitation is followed by resuspension. Precipitation and resuspension should be avoided if the 2-D experiment focuses on the complete and accurate representation of all the proteins in a sample. The different methods by which the proteins can be precipitated are:
- Ammonium sulphate precipitation (“Salting out”)
- TCA precipitation
- Acetone precipitation
- Precipitation with TCA in acetone
- Precipitation with ammonium acetate in methanol
The decision to employ these steps depends on the nature of the sample and the experimental goal. Complete solubilisation and denaturation must be done prior to first-dimension IEF. Complete denaturation ensures that each protein is present in only one configuration, and that aggregation and intermolecular interaction is avoided.
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2-D Electrophoresis using immobilized pH gradients: Principles and Methods, Amersham Biosciences, 80-6429-60, Edition AC