By Alexander E. Kalyuzhny
This moment variation quantity presents precise protocols that handle the demanding situations of signal-transduction IHC. This ebook delves into chapters that debate the character of sign transduction phenomena and methods to creating phosphor-specific antibodies, in addition to a number of bona fide equipment tools on electronic imaging strategies, maintenance of tissue objectives, multicolor detection, circulate cytometry, lipophagy research, apoptosis, and the mix of IHC with in situ hybridization. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and heading off identified pitfalls.
Thorough and entire, Signal Transduction Immunohistochemistry: equipment and Protocols, moment version is a necessary source to either newcomers and specialists in different fields of biomedical examine who desire suggestion on IHC protocols to review sign transduction. This publication can be worthy for researchers in academia, govt labs, and the biotech industry.
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Additional info for Signal Transduction Immunohistochemistry: Methods and Protocols
Recently, multiplexed phospho-flow has elevated the understanding of complex signaling abnormalities in cells from AML, Lupus, and other diseases [101, 102]. Phospho-flow has proven useful in the analysis of phosphorylation networks of key proteins like STATs, ERK1/2, and p38 MAPK in cells from AML patients. In unique cell populations, it has been possible to stratify AML patients according to their responsiveness to specific ligands. It has been shown that individual patients have unique differences in signaling pathways at the cellular level that may correlate with resistance or response to some therapeutics.
Many proteins are assembled into oligomeric complexes and their signal may land in a nonspecific spot, requiring unique lysis buffer conditions to break down these large protein assemblies. As with the previously discussed approaches, once a signal, or lack thereof, is observed with an antibody array, it is advisable to confirm it by other methods before proceeding to a large scale study. Nonetheless, the availability of antibody arrays has helped scientists obtain answers in less time than before.
Second, the affinity of each antibody needs to be high enough to collect all of the protein in a sample from a cell extract or fluid. Due to the limiting and transient nature of phosphorylation events, in our experience, the single antibody approach often does not provide a robust and reproducible fluorescent signal from total cell extracts. It is estimated that the lower limit of sensitivity of a fluorochrome is about 1 ng/mL, which may not nearly sensitive enough to detect events of low frequency such as most phosphorylations.