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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