The transition temperature of ELPs has been shown previously to depend linearly on NaCl concentration. In our initial experiments in buffered conditions, identical Tt were observed at 15 mM and 150 mM NaCl. However, in the absence of buffer, we observed a decrease in Tt with increasing NaCl as expected (Figure ). With increased KI8 concentration (1 mg/mL), the sensitivity of the Tt to salt concentration increased further. This is consistent with the hypothesis that salt is playing a dual role in lowering the Tt of KI8, both by increasing solvent polarity and reducing charge-charge repulsion between polymers by electrostatic screening. KI8 also had a lower Tt without buffer at both NaCl concentrations. Taken together with the I90 results, these results further provoked us to investigate the hypothesis that there may be buffer-specific interactions with KI8 contributing to its increased solubility even in the presence of NaCl. HEPES has previously been observed to adsorb onto protein surfaces, influencing their phase separation behavior and noting a weakly attractive, "salting-in" effect. Supporting the hypothesis that this phenomenon is occurring for KI8, we observed a reduction in surface charge for KI8 below the Tt in the presence of 10 mM HEPES as reflected by zeta potential measurements (Figure ). This result is consistent with the interpretation that HEPES buffer molecules adsorb onto individual KI8 monomers, modifying their electrophoretic mobility and thus their measured zeta potential. When protein -protein interactions become stronger than protein -buffer interactions (i.e., above the Tt), the zeta potential of the phase-separated KI8 particles now follows the same temperature trend as the water solution. In the presence of NaCl, a strong electrolyte, the surface charge on KI8 is further reduced, in this case including after phase separation, due to general electrostatic screening and competition for adsorption at the protein surface. Evidence of the competition between salt and buffer ions for protein surface binding is seen in Figure ; in the presence of competing buffer molecules, the influence of NaCl on KI8 LCST is reduced. We hypothesize that the "salting-in" effect of HEPES is at least somewhat dependent on the charged state of Lys side chains, as it has been previously demonstrated that buffer molecules can specifically adsorb to charged protein surfaces. Supporting this, the Tt of KI8 in 10 mM HEPES pH = 11.8 (20 °C) is closer to the Tt of KI8 in deionized water (18 °C) than in HEPES pH = 7.4 (28 °C) (Figure ). However, HEPES may also weakly associate with the hydrophobic surfaces of KI8, a proposed mechanism for the salting-in effects observed with chaotropes, given that the Tt is slightly elevated by HEPES even at high pH. Reported small molecule modulators of phase separation include bis-ANS and Congo red, both of which, like HEPES, contain negatively charged sulfonate groups (Figure ). We performed temperature-ramp experiments on KI8 at various concentrations of HEPES, all at pH = 7.4 (Figure ). We observed an inverted U-shape dependence of Tt on HEPES concentration, similar to previously observed nonlinear effects of the chaotropic ions SCN -and I - on ELP and pNIPAM LCST behavior. HEPES at pH = 7.0 exhibits positive Jones-Dole B viscosity coefficient values, indicating a kosmotropic or "water-structuring" character. Our model system suggests that at low (< 10 mM) concentrations, HEPES is predominantly acting by weakly associating with KI8. This weak association has multiple effects: it will decrease surface charge, increase conformational stability, and directly compete with protein -protein binding.