It is known that composition and concentration of the participating macromolecules, the flexibility of these macromolecules, and environmental factors such as pH, temperature, and ion concentration affect phase separation. In particular, multivalent interactions between macromolecules, such as electrostatic interactions between residues in IDPs, appears to be essential for phase separation; at the molecular level, temporary electrostatic interactions between oppositely charged polyelectrolytes driven by valency can also result in phase separation. For example, phase separation of IDRs in Ddx4, an RNA-binding protein found in germ granules, has been attributed to electrostatic interactions between clusters of opposing charges and cation-pi interactions between FG and RG motifs. Additionally, the mitotic spindle protein BugZ and the RNA-binding proteins FUS, hnRNPA1, and hnRNPA2 phase separate due to Tyr-Arg interactions. When considering valency effects on phase separation caused by binding of two macromolecules (considering the most simplistic case), the following factors must be considered: a) structural valency, i.e., the number of binding sites available per molecule, b) effective valency, i.e., the number of sites that are sterically, simultaneously accessible for intermolecular contacts, and c) interaction energy, i.e., the affinity between binding sites on each molecule. For a two-component system made up of associative polymers, a "sticker and spacer" model, which considers all of these factors, can be used to understand how multivalent interactions drive phase separation. In this model, a polymer contains "sticker" groups along the chain that can bind reversibly with other sticker groups (on the same or a different polymer) through weak chemical or physical interactions such as H bonds, electrostatic interactions, hydrophobic interactions, etc. Spacers are the regions between stickers, and enable cross-links by physically bringing stickers together (Fig. ). With the help of spacers, the physical crosslinks between stickers on two interacting associative polymers can result in a density transition (also known as phase separation), whereby when both polymers are above a threshold concentration (csat), a polymer-dilute phase (comprising minimal intersticker crosslinks) and a polymer-dense phase (containing crosslinked stickers) will form. This model requires two specific assumptions. First, that interactions between spacers, such as those between stickers and spacers, spacers and other spacers, and spacers and solvent, balance one another out and cause the spacer areas to operate as an ideal chain. Second, that after phase separation, the polymers themselves have similar structural preferences regardless of which phase they are in, ruling out the idea that conformational changes due the physical/chemical properties of different phases could affect the valence of stickers (thus keeping all valence-related variables constant). To illustrate the usefulness of the sticker and spacer model, let us examine one case study in which the sticker and spacer model was used to demonstrate the mechanism of phase separation. The CT-10 regulator of kinase II (CrkII), a signaling adaptor protein, identifies proline-rich motifs (PRMs) in binding partners like cAbl kinase through its N-terminal Src homology 3 (nSH3) domain. Synthetic SH3/PRM system repetitions have thus been used to clarify the role of these interacting domains in promoting phase separation and are particularly of interest because of their role in cancer metastasis, cell spreading, and microbial pathogenesis. Within this model, stickers on SH3 thus interact with stickers on PRM (Fig. ). These cross-links have a finite time span (making and breaking of these bonds/interactions) based on interaction strength, range, and directionality between these domains, and the timescale of the making and breaking these cross-links and the valency of the participating polymers determines the physical properties and dynamics of the system itself. For example, SH3 and PRM constructs with higher valency (higher number of binding sites) form networks/cross-link faster, resulting in more rapid phase separation. If SH3 and PRM are mixed at equimolar concentration, gradually increasing their concentrations at an identical rate of time results in more sticker-sticker interactions between SH3 and PRM. When the concentrations of both SH3 and PRM exceed csat, phase separation occurs, resulting in separation of the polymers into a dense phase and a dilute phase. Within the dense phase, we observe a networked system of PRM and SH3, bound together by sticker-sticker interactions and highly concentrated in both PRM and SH3 (cdense). However, in the dilute phase, the absence of sticker-sticker interactions results in an unnetworked system and a lower concentration of PRM and SH3 (cdilute) (Fig. ).