Interactions with MEK and ERK which are present in much higher concentration than RAF allows signal amplification. Removal of the phosphate from RAF causes a return to its baseline isoelectric point and rapid relocation back to the cell membrane where it is available for subsequent signal transduction. The expected steady state distribution of messenger proteins in the MAPK proteins was predicted using a purely diffusion dynamics and the proposed IEFM. Experimental observations in HMEC cells were consistent with the IEFM predictions. The predicted rapid movement of the MAPK proteins could not be experimentally observed. For this reason, we examined the computation models assuming a steady state with continuous presence of ligand at the CM as would be expected under normal culture conditions. To better capture the full biological dynamics, we added interactions with the scaffolding protein KSR1. Scaffold proteins have been found to play a large role in modulating the signaling strength and regulating the signal amplitude and duration of the MAPK pathway. The overall role of these scaffold proteins is currently under investigation. KSR1 is one of several such proteins that mediate MAPK protein movements but it is well described and its addition to the model seemed reasonable. For our simulations, we assumed that MEK was usually bound to KSR1 and that this complex interacts with pRAF and ERK permitting the sequence of phosphorylations that result in formation of pERK which then unbinds from KSR1 and moves toward the NM. We also assumed that pERK can bind at a separate site on KSR1 preventing additional pRAF binding. We modeled these interactions under two scenarios. First, we assumed that no intracellular field was present and examined the expected distribution of proteins with movements governed purely by random walk. Second, we assumed the presence of an intracellular electric field and intracellular pH gradient, with protein localization and movement governed by these physical properties interacting with proteins based on their size and isoelectric point. The cytoplasmic pH was assumed to range from 7.2 near the nucleus to 7.4 in the peripheral cytoplasm based on experimental PR-957 measurements. These values are consistent with published reports. In each case, we simulated the expected location of unphosphorylated RAF, MEK and ERK. Changing the pH of the cytoplasm will change the distribution of both the unphosphorylated and phosphorylated messenger proteins by their isoelectric points. This phenomena can be compared to performing isoelectric focusing technique on IPG gels with different pH ranges. For example, if the cytosolic pH drops dramatically then a different set of proteins with lower pI’s will be separated out instead of the ones separated by a normal pH. This could be interesting to study what pH ranges of different pathways result in the most efficient information transfer for that pathway. For example, it is possible to negate the MAPK pathway dynamics if the pH drops below 6.0 in the cytoplasm. In this way the RAF, MEK, and ERK, would all be pushed to the cell wall, breaking down the organization and efficiency. This example seems extreme but it is easy to imagine that some pathways take a much less drastic change in pH to affect.