Electrophoretic pattern

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BioCentrum provides analysis of proteins on the basis of elecrophoretic techniques such as SDS-PAGE under native and denaturing conditions, isoelectric focusing (IEF) and 2-dimensional electrophoresis (2DE). Electrophoretic analyses are performed in mini-sized as well as in large-format gels (or pH gradient strips for IEF). Brilliant Coomassie Blue (CBB) or Amino Black are used to visualize gels. The sensitivity of that detection allows the identification of 1 µg protein in the band. For the detection of 10 ng protein in band, silver staining is performed, which is much more sensitive method.
For quantitative studies, densitometry analysis of gels/membranes, or their electronically processed images, are performed.

Western Blot (WB). Furthermore, BioCentrum offers Wester-Blot (WB) analysis, where the electrophoretically separated proteins are transferred to a membrane (nitrocellulose or PVDF) followed by immunodetection.

SDS-PAGE in denaturing conditions. This type of electrophoresis provides easy and accurate determination of molecular weights of separated proteins. The vast majority of the proteins, especially after the reduction of disulphide bridges, is soluble in electrolytes containing SDS. Separation of proteins takes place according to their molecular weight, because samples have identical charge per unit mass due to binding to SDS. Staining of protein-SDS complexes are much more efficient than only protein. Moreover, the presence of SDS effectively eliminates the enzymatic degradation of proteins during separation.

Native electrophoresis is a type of electrophoresis where native proteins are separated in polyacrylamide gels. The advantage of this electrophoresis in native conditions is the analysis of protein complexes, which in denaturing conditions would be degraded. However, the disadvantage of this method is the relatively poor resolution.

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for protein separation on commercially available strips with immobilized aliphatic ampholytes of different pH values. Amphoteric substances (proteins, peptides) migrate toward the electrodes with opposite sign of cargo in an electric field. In the point, where pH of electrolyte is equal to pI of macromolecule, the resultant charge of this macromolecule is zero. As a result, electrically neutral protein/peptide stops and the separation is obtained according to its pI value.

Two dimensional gel electrophoresis (abbreviated as 2-D electrophoresis or 2-DE) is a method of gel electrophoresis which is commonly used to separate or analyze mixtures of many proteins, such as the complete set of proteins (proteome), present in a given cell populations. 2-D gel electrophoresis separates proteins in two dimensions, according to its isoelectric points and molecular mass. This allows to separate proteins that otherwise could not be separated or distinguished on 1-D gels. Typically, for 2DE analysis on mini gels 10-100 ug of protein is required when silver staining method is used, and 200-500 ug for Coomassie Blue staining. For big gel format 100-300 ug is required when detection is made by silver staining and 1-3 mg for Coomassie Blue staining.


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