Biological membranes are essential for varied cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. remedy (Aveyard and Haydon, 1973). As Eq. 1 shows, the mobility of charged particles in an external electric field is definitely directly related to the magnitude of and therefore move with higher velocity in a given electric field. Whereas optical electrophoresis measurements provide an unambiguous measure of electrokinetic mobility and zeta potential, translating these measurements into info on proton dissociation characteristics of titratable organizations requires more detailed evaluation. This protocol clarifies the preparation of appropriate model membranes, measurements Rabbit Polyclonal to BLNK (phospho-Tyr84) of zeta potential using optical electrophoresis, and data analysis using Gouy-Chapman-Stern formalism to obtain lipid pis either the total formal charge or the elementary charge buy BB-94 ( 1.6022 10?19 C) within the ionized titratable group, is definitely a constant for a given lipid system (Notice 9). The effective charge denseness of lipid headgroups (requires the form: (is the valence of the relevant varieties, is the Faraday constant (9.649 104 C mol?1), is the common gas constant (8.314 J mol?1 K?1), is the complete temp (K), is quantified while = +1) and a decrease in anions (= ?1) at distances approaching the negatively charged bilayer surface. The Gouy-Chapman equation, based on the Poisson-Boltzmann relationship, relates surface potential (is the bulk concentration of ion of valence and buy BB-94 (see Figure 2). To quantify as a function of distance from the surface, we first consider the Debye constant, (m?1), quantified as based on these relationships, we must make an assumption about the distance at which the shear plane exists such that for each measured value of zeta potential, we can assume that = measurements over a range of solution pH values are analyzed to evaluate the pin this example are as follows: pH 1, = ?6.899 mV; pH 2, = ?27.273 mV; pH 3, = ?57.717 mV pH 4, = ?88.171 mV; pH 5, = ?110.527 mV; pH 6, = ?118.836 mV pH 7, = ?120.118 mV; pH 8, = ?120.255 mV; pH 9, = ?120.268 mV Step 1 1: Enter experiment parameters The first column relates to characteristics of the model lipid system: Cell C4 (data input): This is the formal charge per ionized lipid headgroup. POPG is an anionic lipid with a maximum of one negative charge per lipid; therefore, a value of ?1 is input here. Cell C5 (data input): This is the mol% anionic lipid in the model system. In this example, liposomes are pure POPG (in terms of total charge per ?2. Cell C7 (formula input): This recasts buy BB-94 in terms of C m?2 for use in the adsorption isotherm for direct comparison with the Gouy-Chapman equation. The second column relates to the ionic characteristics of the bathing solution: Cell H3 (data input): This is the molar concentration of monovalent electrolyte in the solution. In this example, the solution contains 10 mM NaCl; therefore, a value of 0.01 is entered here. Cell H4 (formula input): This is the formula input for the Debye constant (Eq. 6), given that |z| = 1 and assuming that in terms of ?. Cell H7 (data input): This is the assumed distance x of the slip plane from the surface of the bilayer (correlating with the point at which and x, which is used in Eq. 7. Step 2 2: Calculate surface potential For each pH-specific value of (V) for each pH condition are entered. Cells D13CD21 (formula input): This is a rearrangement of Eq. 7 that is used as the objective cell in the Solver protocol to calculate using Solver, open Solver (from the Excel Tools dropdown menu) and perform the following for each data point: Set objective for the relevant Eq. 7 cell (based on the known concentration of electrolytes in solution and the calculated values of em /em 0. Cells H13CH21 (formula input): This is the calculation of the surface H+ concentration based on the Boltzmann distribution (Eq. 4a). Cells I13CI21 (formula input): This is the calculation of the surface cation concentration based on the Boltzmann buy BB-94 distribution (Eq. 4b). Cells J13CJ21 (formula input): This is an algebraic manipulation of Eq. 3, which solves for em K /em a assuming that the association constant of cation adsorption to the POPG headgroup is 0.6 M?1. Cell J22 (formula input): This takes the average em K /em a value for all measurements and converts it to a p em K /em a value. Open in a separate window Figure 4 Excel sheet workup for data analysisA. Data entry cells, subdivided into steps 1C3. Cells in green are those used for direct data input. Non-shaded cells are those either containing formulas or calculated values. See text for detailed explanation..