Experimental Programs For Silac In Protein Interaction-tamiflu

Meditation Identifying MS and analyzing protein quantification is the main theory of this technology to judge the specific binding protein bait . When certain protein content in the experimental group and control group reaches the statistical difference, it can be sure that the bait proteins interact with proteins. Program one Taking normal cell line as experimental material. 1.Culturing cells with light and heavy chain amino respectively. When it is cultured to five and six generations, the same amount of cells should be mixed and proteins also should be extracted from it. 2.Extracting the light and heavy chains proteins, which should be processed for IP technology with the help of specific antibodies of normal IgG and anti-bait proteins. 3.Mixing the IP products of light and heavy chain proteins, which then should be separated by SDS-PAGE technique. 4.Plastic inner digestion, peptide extraction, purification, identification and quantification. Program two Using over-express bait protein cell lines as experimental material. 1.Culturing cell lines(with labels) of normal cells and over-expressed bait protein with light and heavy chain amino acid. When it is cultured to five and six generations, the same amount of cells have to be mixed and the proteins can be extractd from it. 2. The light chain and the heavy chain of the total protein cells extracted should be in processed of IP with the help of tag protein antibodies. 3. Mixing the IP products of light and heavy chain proteins and then it will bw separated by SDS-PAGE. 4. Plastic inner digestion, peptide extraction, purification, identification and quantification. Program three Using cell lines of gene RNAi bait as experimental material. 1.Using the light chain and heavy chain amino acid to culture cell lines and control cell lines of RNAi bait genes for five and six generations, and then extracting proteins. 2.Extracting the light chain, the heavy chain of the total cell protein and using the specific antibodies, which are against the protein bait, for IP. 3.Mixing the IP products of light and heavy chain proteins and then it is separated by SDS-PAGE. 4. Plastic inner digestion, peptide extraction, purification, identification and quantification. Advantages of using SILAC .pared with traditional CoIP or pull down, SILAC has strong specificity, low false positive pole, a high-throughput, LC-MS and large limitation, as well as the detection of the relative content of other proteins, which can interact with the bait protein. Whats more, when .pared with vitro labeling technique, SILAC can detect and quantify thousands of proteins accurately and it needs fewer protein samples. SILAC is a vivo labeling technique, which will not affect cell functions. So it is widely used and wel.e in the SILAC proteomics analysis field. About the Author: 相关的主题文章: