信号通路研究相关技术(WB、IP、IHC、IF)

What is a Western Blot?
Proteins from cells are separated based on their size using an electric current. An antibody tΒιβλιοθήκη Baidu the protein of interest is used to determine whether that protein is present in the cells.
Cell Signaling Technology® Antibody Validation
• Verifying Specificity And Reproducibility ensures that the antibody performs consistently in all applications specified. • Analysis of a large panel of cell lines with known target expression levels confirms target specificity • Treatment of cells with appropriate kinase-specific activators and/or inhibitors verifies specificity • Phosphatase treatment to verifies phospho-specificity • Correct subcellular localization or treatment-induced translocation verifies target specificity • Comparison of antibody to isotype control ensures acceptable signal-to-noise ratio • Target-specific signal verified in transfected cells, knockout cells, or siRNA-treated cells • Blocking with antigen peptide confirms elimination of specific signal • Side-by-side comparison of a new lot with previous lots ensures lotto-lot consistency
Signaling Pathways Research Techniques
Cell Signaling Technology® Antibody Validation
• Testing In A Number Of Applications allows you to choose the antibody that works best in your intended application. • W =Western blot • IP=Immunoprecipitation • IHC=Immunohistochemistry • IF=Immunofluorescence-based assays • F=Flow cytometry • ChIP Chromatin Immunoprecipitation
This is a Western blot. The dark bands show where the antibody has bound to the protein of interest. You can see that the protein is located just above the 37 kDa ―molecular weight marker‖ (the measurements on the left side of the picture). Thus, the ―molecular weight‖ of the protein is approximately 40 kDa.
Cell Signaling Technology® Antibody Validation
• Identifying Optimal Conditions saves your precious time, samples and reagents. • Optimal dilutions and buffers predetermined • Positive and negative control cell extracts specified • Detailed protocols already optimized
Western Blotting Summary
Using Western Blotting To Study Cell Signaling
Cascading Pathways & Networks B A B* C C* D
1. Western Blotting can be used to analyze the expression, and/or modification status of a protein. 2. Signal transduction: the process of activating and coordinating cellular responses to stimuli. 3. Signal transduction can take many forms. 4. A cascade of signals offers numerous point of signal amplification, regulation, and pathway integration. 5. Kinase cascades are common mechanisms whereby phosphorylation of a downstream kinase activates it‘s kinase activity, propagating the signal. 6. Many signaling modifications: cleavage, methylation, acetylation, phosphorylation, ubiquitination, sumolyation, neddylation, farnesylation,…
内容
• Key Points and Troubleshootings of Western Blotting • IP/ChIP • IHC • IF
Key Points and Troubleshootings of Western Blotting
Western Blotting
• What is Western blotting? • Step by step guide to the procedure and important points for optimization • Troubleshooting • Summary (what common errors to look for)