SERVICES OF MONODIMENSIONAL ELECTROPHORESIS
Electrophoresis on polyacrylamide gel (PAGE) is one of the most used techniques for the separation of macromolecules, including DNA, RNA and proteins.
Electrophoresis can be defined as a process in which an electronic field is used to “move” the charged molecules within a solution. In this method the mobility of a charged molecule is directly proportional to its net charge and the resistance of the solution in which it is moving.
The Sodium Dodecyl Sulfate (SDS) is an anionic detergent, and within the pH range in which it is dissolved, its molecules possess a net negative charge. A polypeptide chain connects a certain SDS quantity in proportion to its relative molecular mass, and the negative charge of the SDS will destroy most of the complex structure of the proteins, especially following a reduction process involving beta-mercaptoethanol or Dithiothreitol (DTT). At that point, the proteins completely lose their initial complex structure composed as a clew, and they succeed in “spreading” in such a way that their mobility depends exclusively on their molecular mass or molecular weight. The protein extract which becomes in that way easily analysable, is put on a polyacrylamide gel so that the proteins move according to their apparent molecular weight. In this way they are separated and can also be semiquantitatively analysed. The SDS PAGE is broadly used in proteomic analyses also considering the protein size, its identification, the analysis of its purity, the identification of its disulfide bridges and the quantification of the protein.
Available dyes: colloidal Coomassie, silver dye compatible with Mass Spectrometry
Bioinformatics: quantitative analysis of one-dimensional tires
Control with antibodies: services of immunoblotting on membranes of Nitrocellulose (semidryblot) making use of chemiluminescence and colorimetric methods.
For molecular masses inferior to 10KDa one can use methods for identification of polypeptide molecules (polypeptides/proteins) on tricine gels.
TWO-DIMENSIONAL ELECTROPHORESIS TECHNIQUES
Two-dimensional electrophoresis (2-DE) is widely used method for its resolving power within the analysis of complex protein mixtures extracted from cells, fabrics, or other biological fluids. Thanks to this technique it is possible to separate the proteins on the basis of two independent characteristics used at different times within the process which are the isoelectric point (pI) and the molecular weight (MW). Each spot produced by this two-dimensional array corresponds to a single type of protein within the analysed sample. It allows to separate millions of different proteins, obtaining information regarding the isoelectric point, the molecular weight and relative abundance within the sample.
Available dyes: colloidal Coomassie, silver-nitrite dyes compatible with Mass Spectrometry, Syro Ruby dyes and 2D DIGE (Different in Gel Electrophoresis using cyanine) analysis
Image analysis using software dedicated to the subject, including analysis of statistical differences
Bioinformatic analysis: String, Panther