What you need to know about protein analysis

  • Gel electrophoresis is an important method for protein analysis, including sds-page and IEF. Sds-page, which is used to analyze the most commonly used protein gel electrophoresis, provides a simple way to estimate the molecular weight of the protein, evaluate the complexity of the sample or the purity of the preparation, and monitor the fractions obtained during chromatographic or other purification processes. The electrophoresis method has been used in protein research for many years. With the wider range on protection of new plant varieties in China, electrophoresis technology will play a more important role in variety identification.

     

    Western Blot is a common method of protein analysis, such as qualitative analysis and semi-quantitative analysis. In this method, proteins are separated by 2-de and then transferred to the carrier membrane. Western blot can be used for qualitative detection of monoprotein and protein modification.

     

    Here's the intro:

     

    Two-dimensional gel electrophoresis (2-de) is a commonly used gel electrophoresis technique, and 2-de is a powerful tool for the separation and separation of complex protein mixtures for the analysis of protein mixtures in two-dimensional space. In two-dimensional gel electrophoresis, protein was separated by pI in isoelectric focusing, and then further separated by molecular weight through sds-page, and the sample protein was distributed in two-dimensional gel profile. The technology expands the number of identifiable proteins, providing more effective data and detailed information for protein analysis. The most demanding part of two-dimensional electrophoresis is image comparison and automatic analysis. In order to evaluate and compare complex two-dimensional graphics, gel imaging must be converted into digital data by a scanner or camera, and further analysis by computer software.

     

    Western Blot was used to detect specific proteins in tissue homogenate or extract samples. It uses gel electrophoresis to isolate the three-dimensional structure of natural proteins, or the polypeptide length of denatured proteins. The isolated proteins are then transferred to a membrane (typically nitrocellulose or PVDF) where they are stained with antibodies specific to the target protein. Western Blot is widely used in molecular biology, biochemistry and immunology. Imprinting is the transfer of macromolecules to fixed membranes for the detection of specificity and sensitivity. In Western Blot analysis, the protein of the sample was first isolated by means of isoelectric point (pI), molecular weight, electric charge in gel electrophoresis, or by multiple methods such as sds-page and IEF. After gel electrophoresis, the protein is transferred from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), making it easy for antibody detection. The binding capacity of PVDF membrane to protein is higher than that of nitrocellulose, but nitrocellulose can better bind to small protein. The main method of transferring the protein is called electrophoresis, which USES an electric current to pull the protein from the gel to PVDF or nitrocellulose membranes.

    Western Blotting can be used to track protein phosphorylation. Antibodies bound to all subtypes of the polyphosphorylated protein will show a "" string of beads" "appearance on the 2D gel. Western Blotting also helps to identify known and unknown proteins in the complex produced by immunoprecipitation. Once the protein of interest is located on the Western blot, the corresponding spots can be cut from the Coomassie blue dye gel and identified by MS.

     

    The process of protein gel analysis and gel image analysis is complex, but with the development of mass spectrometry, protein and peptide identification is no longer a tedious and time-consuming work.In order to shorten the time of protein identification and improve the accuracy of protein sequencing, the mass spectrometry technology platform has been greatly improved. Up to now, a comprehensive protein identification pipeline has been developed, from sample preparation, protein isolation and purification, quality analysis to systematic bioinformatics analysis. Although protein identification is no longer a problem, mastering the analysis of proteins is still of great importance to scientific research and deserves our attention!