This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other. The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional". The cookie is used to store the user consent for the cookies in the category "Analytics". These cookies ensure basic functionalities and security features of the website, anonymously. Necessary cookies are absolutely essential for the website to function properly. The right combination of antibodies targeted to strategic epitopes will bring clarity to the state of any intracellular signaling cascade. The antibody selection guide will lead you to the best options for single-purpose antibodies or those that you will use in multiple applications. It takes into consideration the specimen species, the host species, the desired level of specificity, and cross-reactivity concerns, among other factors. Our antibody selection guide makes picking the right combination of antibodies easy. Single domain antibodies are also useful for these experiments, which add to the versatility of combinations of antibodies that can be used in the same assay.īoth monoclonal and polyclonal antibodies are useful in detecting the state of intracellular signaling cascades. It is thus important that the right host species for primary and secondary antibodies are considered with forethought. Multiple epitopes, whether on the same protein or different proteins, can be simultaneously or sequentially detected on the same blot or same cells. Since a protein can have multiple phosphorylation sites, each of which affects the protein differently, in addition to multiple sites for moieties such as ubiquitination, picking the right epitopes to detect is a fruitful – but nontrivial – task. Phosphorylated epitopes are often targets of immunoblotting and in situ immunolocalization assays. Thus, antibodies that are designed to detect the presence or absence of a chemical moiety on a signaling protein can give researchers a clear picture of what pathway has been activated and to what extent. Each active conformation of a protein or phosphorylation site can serve as a different epitope that can be bound by an antibody. The aforementioned complexity of intracellular signaling is precisely why picking the right antibodies is crucial when researchers seek to accurately determine the state of a cell in response to extracellular stimuli. Picking the Right Epitopes for Accurate Information The ability of scaffold proteins to bind MEK and ERK is also regulated in terms of the scaffold’s activation state, which changes after post-translational modification by the addition of chemical moieties. Proteins such as MEK and ERK are known to bind near each other on scaffold proteins. For example, phosphorylated MEK or ERK is often interpreted as representing activation of an intracellular signaling pathway downstream of a GPCR.įurthermore, these signaling proteins can have multiple sites of phosphorylation, different combinations of which affect the level of the protein’s activity or half-life. The activation state of a signaling protein is often used as an indicator of whether a certain pathway has been activated. They also depend on the presence or absence of moieties, such as phosphate groups, ubiquitin groups, and calcium ions. These states depend on conformational changes that result from interactions with other proteins. The complexity of intracellular signaling is that the proteins in a signaling cascade have varying states of activity (i.e. Activation States of the Signaling Cascade
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