Bidirectional Gel Electrophoresis (2-DE)

the principle of
bidirectional gel electrophoresis is that the first isoelectroric point based on
protein
isoelectratic focus separation, and the second direction is separated by SDS
-PAGE according to the molecular weight, separating the proteins in the complex protein mixture on a two-dimensional plane. In recent years, many improvements have become the most valuable core method for studying proteomics.The two key parameters for separating all proteins in proteomics are their resolution and repeatability. Under the present circumstances, a piece of plywood (16cm × 20cm ) for two-way gel electrophoresis can be divided into 34A thousand, 000 detectable protein spots, which is still too few compared to the 10 million
gene
expressable proteins.the1980s began to use fixed pHgradient glue, overcoming many shortcomings such as carrier sex electrolyte cathode drift and so on to establish a very stable and easily set pHgradient. Since a very narrow pHrange (e.g. 0.05U/cm)can be established, areas of particular interest can be analyzed for a second round in a narrow pHrange, which greatly improves resolution. This kind of glue strip already has the commodity production, so basically solves the problem of two-way gel electrophoresis repetition. This is a very important breakthrough in two-way gel electrophoresis technology. The second-way SDS-PAGEhas vertical plate electrophoresis and horizontal ultra-thin plastic electrophoresis, which can separate 10to 100kDmolecular weight proteins.the highly sensitive silver staining method detects 4ngproteins, the most sensitive of which is labeled with
isotopes
, and the 20ppm marker protein can be measured by its fluorescence or phosphorescent intensity. Using image scanner, Lacey density meter, charge combination device can digitize the protein map obtained by the above method, and then after computer processing, remove the vertical and horizontal tail and background background color, you can give the exact location and intensity of all protein spots, get the image full of protein spots, that is, the so-called "reference glue map". The main difficulty in proteomics research is the qualitative and quantitative analysis of proteins isolated by electrophoresis with bidirectional gels. The most common method is to print the proteins on the glue to the PVDF (polyvinylidene difluoride membrane before analyzing them to determine whether they are known or unknown. Now the grading analysis method is to do a fast
amino acid
composition analysis, but also can do 4 to 5 cycles of N end trace sequencing, and then do amino acid composition analysis; It has been reported data 4 at the end of the N-base can give a lot of information and get fairly accurate results. If combined with C sequence, the accuracy of the judgment results will be higher. Further identification of the isolated protein requires a sufficient amount of pure protein, which has been highly purified during electrophoresis. A piece of plywood now allows samples up to mg orders of magnitude, so each isolated protein spot can have a μ g amount of protein, so that otherwise trace amounts of protein can also be identified. The translation and modification and processing of proteins refers to chemical reactions carried out after the synthesis of peptide chains, such as
phosphorylation
, hydroxylation, glycosylation, desulfur bond formation, and the recently discovered self-shearing of proteins, etc., there may be more than 100 kinds. Post-translation modification and processing are necessary for the normal physiological function of proteins, and their changes are often related to the occurrence of disease. Electrophoresis with bidirectional gels can be used for post-translation modification studies, such as 32P markers can be used to study changes in phosphorylation proteins. Protein towing, which is often found in bidirectional gel electrophoresis, is most likely caused by different translated modification products of a protein. Dragging image changes may provide important information on disease diagnosis. current challenge with bidirectional gel electrophoresis technology is: (1) of low-copy proteins. The body's trace proteins are often important regulatory proteins. In addition to the method of increasing the sensitivity of bidirectional gel electrophoresis, the most promising is to use media-assisted laser de-absorption / ionization
mass spectrometry
on PVDF membranes, but the current technology is not enough to detect proteins with fewer than 1000 copies. (2) the separation of polar acids or alkaloid proteins. (3) (> 200kD ) or very small (< 10kD the separation of the protein. (4) insoluble proteins, which include some important membrane proteins. (5) high-quality bidirectional gel electrophoresis requires excellent technology, so there is an urgent need for automatic two-dimensional electrophoresis.