Two-dimensional gel electrophoresis

Isoelectric concentration can be combined with SDS-PAGE to acquire very high resolution separations in a process which is known as two-dimensional gel electrophoresis. Firstly the protein sample is subjected to isoelectric focusing in a narrow strip of gel containing polyampholytes. This isoelectric  focusing  gel strip is then  placed   on  top  of  an  SDS-polyacrylamide  gel  and  electrophoresed   to

Figure: SDS-PAGE. (a)  Appearance of proteins after electrophoresis on an SDS polyacrylamide gel. Lane 1, proteins (markers) of known molecular mass; lane 2, unpuri?ed mixture of proteins; lane 3, partially puri?ed protein; lane 4, protein puri?ed to apparent homogeneity; (b) determination of the molecular mass of an unknown protein by comparison of its electrophoretic mobility (distance migrated) with those of proteins (markers) of known molecular mass.

Figure: Isoelectric focusing.  (a) Before applying an electric current. (b) After applying an electric current the proteins migrate to a position at which their net charge is zero (isoelectric point, pI).

generate  a two-dimensional  pattern  of spots  in that  the  proteins  have  been separated  in  the  horizontal  direction  on  the  basis  of  their  polyacrylamide  and  in  the vertical  direction  on the basis of their mass. The whole result is in which proteins are separated both on the basis of their size and their charge. Therefore two proteins  which have very same  or identical  polyacrylamides, and generate  a single band through isoelectric  focusing,  if they  have  various  molecular  masses  will produce  two spots  through  two-dimensional   gel  electrophoresis.  Equally, proteins with same or identical molecular masses, that would produce a single band through SDS-PAGE, will also produce two spots if they have several polyacrylamides because of the initial separation via isoelectric focusing.  The high resolution  division  of proteins in a complex mixture that can be achieved through two-dimensional  gel electrophoresis  makes this method  extremely  useful for comparing  the proteome the entire complement of proteins in a organism or cell of cells or tissues under several conditions, example for differentiated  versus undifferentiated  cells and is frequently used prior to the analysis of individual protein spots through mass spectrometry .

Fig: Two-dimensional gel electrophoresis. The protein sample is ?rst subjected to isoelectric focusing in one dimension and then to SDS-PAGE in the second dimension.