Aggregation of alkyltrimethylammonium ions at the cleaved muscovite mica-water interface: a Monte Carlo study

The precise molecular structure of organically modified mineral surfaces is still not well understood. To establish a relation between experimental observations and underlying molecular structure, we performed Monte Carlo simulations of the aggregation behavior of alkyltrimethylammonium surfactants (C(n)TMA(+)) at the interface between C(n)TMACl solution and cleaved K(+)-muscovite. The structures were examined with regard to the influence of varying alkyl chain length n (n = 8, 12, 16) and surface coverage of C(n)TMA(+) ions. The simulation results indicate that the water film structure at the muscovite surface is considerably influenced by the adsorption of C(n)TMA(+). A fraction of the C(n)TMA(+) ions forms inner-sphere and outer-sphere adsorption complexes with nitrogen-surface distances of 3.3-3.8 and 5.5-8.4 ?, respectively. The simulated monolayer aggregates exhibit thicknesses of 31-35, 22-27, and ～18 ? for C(16)TMA(+), C(12)TMA(+), and C(8)TMA(+), respectively. C(16)TMA(+) and C(12)TMA(+) ions form bilayer aggregates, which show a strong interdigitation of the two opposing layers composing them. The aggregate thicknesses equal 35-39 and 30-35 ?, respectively, and are in agreement with available experimental data. In contrast, the short-chained C(8)TMA(+) ions do not form bilayer aggregates. In agreement with previous experimental studies, the alkyl chains of the aggregated ions show high conformational order markedly decreasing with decreasing chain length. We suggest that the simulated structures represent C(n)TMA(+) aggregates, which are formed on muscovite during the experimentally observed initial equilibration phase characterized by the presence of inorganic ions within the aggregates.     

Methods development for Analysis of Partially Deglycosylated Proteins and Application to an HIV Envelope Protein Vaccine Candidate

The work presented herein describes the first comprehensive analysis of a partially deglycosylated HIV vaccine candidate envelope protein (Env). The Env, JRFL gp140 ΔCF, with 27 potential glycosylation sites, was partially deglycosylated with PNGase F as part of a strategy to generate a more immunogenic HIV vaccine, and the resulting protein's glycosylation was characterized in a unique workflow using two different glycosidases, Endo H and Endo F3. This unique analysis protocol provided for coverage on 26 of the 27 glycosylation sites, and the data showed that the biochemical treatment with PNGase F resulted in a highly heterogeneous glycoprotein product that had been partially deglycosylated at most of the glycosylation sites. The protocols described in this work could be useful for characterizing the glycosylation site occupancy of other native or biochemically deglycosylated proteins.     

载液组成对于卵白蛋白在非对称流场流分离通道中膜吸附和聚集行为的影响

非对称流场流分离技术对于蛋白质等生物大分子的分析具有温和、分离范围广的特点。然而,在场流分离通道中,受载液组成的影响而产生的蛋白质与通道膜的相互作用和蛋白质在通道内的聚集行为,会影响分析物的回收率和尺寸形态,这些现象一定程度上限制了场流分离仪器的进一步应用。该文研究了载液组成对于卵白蛋白在非对称流场流分离中膜吸附和聚集行为的影响。考察了不同pH （6.2、7.4、8.2）、阳离子种类（Na⁺、K⁺、Mg²⁺）及多种离子强度（0~0.1 mol/L）等条件对卵白蛋白洗脱过程的影响。结果表明a）载液的离子强度越大,卵白蛋白的吸附和聚集行为越严重;b） pH和蛋白质的等电点pI的相对大小决定了蛋白质的表面电荷,从而影响蛋白质的吸附聚集行为;c）二价阳离子Mg²⁺更易引发通道中蛋白质的吸附和聚集。这些结果有助于今后使用非对称流场流分离技术分析蛋白质样品时,改善载液组成以获得更高的回收率,降低蛋白质聚集作用,对AF4更广泛地应用于蛋白质生化分析中有较好的参考价值。     

A combined streaming-potential optical reflectometer for studying adsorption at the water/solid surface

A novel in-situ streaming-potential optical reflectometry apparatus (SPOR) was constructed and utilized to probe the molecular architecture of aqueous adsorbates on a negatively charged silica surface. By combining optical reflectometry and electrokinetic streaming potentials, we measure simultaneously the adsorption density, gamma, and zeta potential, zeta, in a rectangular flow cell constructed with one transparent wall. Both dynamic and equilibrium measurements are possible, allowing the study of sorption kinetics and reversibility. Using SPOR, we investigate the adsorption of a classic nonionic surfactant (pentaethylene glycol monododecyl ether, C12E5), a simple cationic surfactant (hexadecyl trimethylammonium bromide, CTAB) of opposite charge to that of the substrate surface, and two cationic polyelectrolytes (poly(2-(dimethylamino)ethyl methacrylate), PDAEMA; (poly(propyl methacrylate) trimethylammonium chloride, MAPTAC). For the polyethylene oxide nonionic surfactant, bilayer adsorption is established above the critical micelle concentration (cmc) both from the adsorption amounts and from the interpretation of the observed zeta potentials. Near adsorption saturation, CTAB also forms bilayer structures on silica. Here, however, we observe a strong charge reversal of the surface. The SPOR data, along with Gouy-Chapman theory, permit assessment of the net ionization fraction of the CTAB bilayer at 10% so that most of the adsorbed CTAB molecules are counterion complexed. The adsorption of both C12E5 and CTAB is reversible. The adsorption of the cationic polymers, however, is completely irreversible to a solvent wash. As with CTAB, both PDAEMA and MAPTAC demonstrate strong charge reversal. For the polyelectrolyte molecules, however, the adsorbed layer is thin and flat. Here also, a Gouy-Chapman analysis shows that less than 20% of the adsorbed layer is ionized. Furthermore, the amount of charge reversal is inversely proportional to the Debye length in agreement with available theory. SPOR provides a new tool for elucidating aqueous adsorbate molecular structure at solid surfaces.     

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (pI) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral pI, myoglobin in this case, while removing other proteins with acidic or basic pIs. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 microg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.     

Ion channels from linear and branched bola-amphiphiles

The synthesis and characterization of the ion channel activity of three new bola-amphiphiles is described. These compounds are conceptually derived from a previously reported bis-cyclophane bola-amphiphile through opening of the cyclophanes to acyclic structures and were found to readily form ion channels in planar bilayer membranes as assessed by bilayer clamp single-channel analysis. All three compounds behaved very similarly: the dominant channels formed by all three are Ohmic with specific conductance of 10 +/- 1 pS (NaCl electrolyte) and 39 +/- 1 pS (CsCl electrolyte). Single-ion permeability ratios, determined from dissymmetric electrolyte experiments, showed the selectivity P(Cs(+)) > P(Na(+)) > P(Cl(-)). Less frequently, lower conductance channels were also observed to act independently of the dominant channels. The lifetimes of the dominant channels range from 70 to 280 ms for the three compounds with some very long-lived openings (20-40 s) observed for two of the three. The lower conductance states have shorter lifetimes. This study demonstrates that bis-macrocyclic compounds are not essential for channel formation by bola-amphiphiles, and opens a new class of channel-forming compounds for structure-activity optimization.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.     

Surface charging of layered double hydroxides during dynamic interactions of anions at the interfaces

In this research, we investigated the effect of dynamic anion adsorption/exchange on the surface charging property of Mg(2)AlClLDH and Mg(2)AlCO(3)LDH particles that show the average zeta potential of 41 and 34 mV in the as-prepared suspension, respectively. The addition of NaCl up to 3x10(-3) M in the suspension does not obviously affect the zeta potential of both LDHs, which can be attributed to the less affinity of Cl(-) to LDH. The introduction of Na(2)CO(3) severely reduces the zeta potential at the CO(3)(2-) concentration higher than 1x10(-4) M, and to the negative value in both LDH systems at ca. 2x10(-3) M, which is presumably resulted from the exchange and the re-orientation of CO(3)(2-) in a tilt/vertical style on the surface. All four organic anions (dodecyl sulfate, folate, citrate and polyacrylate) also significantly affect the zeta potential of the LDH particles. At the lower concentrations of organic anionic groups (<1x10(-4) M), the zeta potential was slightly affected, i.e. limited exchange/adsorption. However, the concentration increasing to some point suddenly decreases and reverses the zeta potential of the LDH particles, which is presumably caused by the hydrophobic interactions that bind the hydrophobic hydrocarbon chains (especially in dodecyl sulfate) into the micelle-like bilayer bunches on the LDH surface. In addition, the effect of pH in 5.5-11.0 on the LDH particle surface charging is mainly reflected through the conversion of CO(3)(2-) to HCO(3)(-)/H(2)CO(3) when pH decreases from ca. 11 to 6, with limited contribution from protonation/deprotonation and exchange/adsorption.     

Protein adsorption in fused-silica and polyacrylamide-coated capillaries

The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.     

Library selection approaches to engineering enhanced retroviral and lentiviral vectors

Retroviral and lentiviral based gene delivery vectors have been used in numerous pre-clinical studies and clinical trials due to their advantages, including stable and prolonged expression of therapeutic transgenes and minimal immune responses against the vector. Despite such advantages, however, retroviral vectors also have several limitations for gene therapy applications. For example, they can suffer from a lack of efficient or targeted gene delivery to key cell types. In addition, retroviral vector stability can be compromised by their envelope proteins. This review briefly describes how such limitations have been overcome by recently developed library selection approaches that borrow a lesson from nature: the ability of evolution to generate biomolecules with novel function. These library selection approaches are based on the construction of retroviral libraries where the sequences encoding natural viral components are partially randomized using a variety of methods in order to generate diverse libraries that can be selected to create improved or novel functions. These high throughput, library-based approaches provide a strong complement to rational engineering of viral components for the rapid development of efficient and safe retroviral and lentiviral vector systems for gene therapy.     

Ion-exchange chromatography of proteins near the isoelectric points.

The retention and the resolution of beta-lactoglobulin A and B (LgA, LgB) were investigated with various ion-exchange chromatography media. The number of sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (=5.1-5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion-exchange chromatography columns where the number of adsorption site values are small (ca. 2-3). The two proteins were not separated on cation-exchange chromatography columns. Factors affecting the resolution and the retention near the pI were discussed.     

Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax glass chips with length 70 mm, 300 microm width and 100 microm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22-5 microg microL(-1). Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes

The effects of adsorption of two kinds of proteins on the membrane characteristics of liposomes were examined at pH 7.4 in terms of adsorption amounts of proteins on liposomes, penetrations of proteins into liposomal bilayer membranes, phase transition temperature, microviscosity and permeability of liposomal bilayer membranes, using positively charged lysozyme (LSZ) and negatively charged bovine serum albumin (BSA) as proteins and negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG) liposomes. The saturated adsorption amount of LSZ was 720 g per mol of liposomal DPPG, while that of BSA was 44 g per mol of liposomal DPPG. The penetration of LSZ into DPPG lipid membranes was greater than that of BSA. The microviscosity in the hydrophobic region of liposomal bilayer membranes increased due to adsorption (penetration) of LSZ or BSA, while the permeability of liposomal bilayer membranes increased. The gel-liquid crystalline phase transition temperature of liposomal bilayer membranes was not affected by adsorption of LSZ or BSA, while the DSC peak area (heat of phase transition) decreased with increasing adsorption amount of LSZ or BSA. It is suggested that boundary DPPG makes no contribution to the phase transition and that boundary DPPG and bulk DPPG are in the phase-separated state, thereby increasing the permeability of liposomal bilayer membranes through adsorption of LSZ or BSA. A possible schematic model for the adsorption of LSZ or BSA on DPPG liposomes was proposed.     

Complex ion effects on polypeptide conformational stability: chloride and sulfate salts of guanidinium and tetrapropylammonium

The effects of chloride and sulfate salts of tetrapropylammonium (TPA(+)) and guanidinium (Gdm(+)) on the conformational stabilities of tryptophan zipper (trpzip) and α-helical (alahel) peptides were measured by circular dichroism spectroscopy. Like Gdm(+), TPA(+) interacts with the planar tryptophan indole group, perturbing the conformational stability of trpzip peptides. TPA(+) effects are largely unaffected by sulfate, indicating an absence of the heteroion pairing that is observed in concentrated Gdm(2)SO(4) solutions. TPA(+) stabilizes helical conformations in alahel peptides, indicating exclusion from the peptide bond. The observations are broadly consistent with predictions of molecular dynamics simulations [Mason, P. E.; et al. J. Phys. Chem. B2009, 113, 3227-3234], indicating that the effects of complex ions on proteins are increasingly predictable in terms of ion hydration, complementary interactions with specific protein groups, and ion-pairing contributions.     

Specific influence of univalent cations on the ionization of alumina-coated TiO2 particles and on the adsorption of poly(acrylic)acid

A surface counterion titration method was used to monitor the interaction of monovalents cations (Li(+), Na(+), TMA(+)) with the surface of alumina-coated TiO(2) particles in concentrated media at different pH and electrolyte concentrations. This method allows measuring separately the negative and positive contribution to the surface charge. It showed that Cl(-) and TMA(+) are indifferent ions, but Li(+) and Na(+) specifically adsorb on the non-ionized alumina surface sites. The binding sequence of cations is Li(+)>Na(+)>TMA(+) at all ionic strengths investigated and is consistent with the structure-making and structure-breaking model developed a few decades ago. Polyacrylic acid (PAA) previously neutralized with the corresponding hydroxide (LiOH, NaOH, TMAOH) has been adsorbed on the alumina surface at different pH. The polymer counterion has a significant influence on the polymer adsorption. The sequence of the surface coverage as a function of the polymer counterion follows the order Li-PAA > Na-PAA > TMA-PAA. The much higher surface coverage with Li-PAA and Na-PAA compared to TMA-PAA is explained by the specific adsorption of Li-PAA and Na-PAA on the nonionized alumina surface sites, the same way LiCl and NaCl do.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.     

Zeta potential of particle bilayers on mica: a streaming potential study

The streaming potential of mica covered by bilayers of latex particles was measured using the parallel-plate channel cell. The size of the first latex (A500) bearing amidine charged groups was 503 nm and the second latex (L800) bearing sulfonate groups was 810 nm (at pH 5.5 and an ionic strength of 10(-2)M). The A500 latex exhibited an isoelectric point at pH 10.5, whereas the L800 latex was strongly negative at all pH. Mica sheets were precovered first by the A500 latex particles under diffusion transport conditions. The coverage of this supporting layer was regulated between 0.02 and 0.5 by changing the bulk concentration of latex and the deposition time. Then, the second layer of the L800 latex of regulated coverage up to 0.55 was deposited under the diffusion transport. The coverage of particles and their distributions in both layers were determined by a direct enumeration of particles by optical microscopy under wet conditions and by AFM. It was shown that the structure of the L800 particle layers and the maximum coverage were in accordance with theoretical simulations performed according to the random sequential adsorption (RSA) model. After forming bilayers of desired composition and structure, streaming potential measurements were carried out. The influence of the mica substrate, the supporting layer coverage, and its zeta potential on the apparent zeta potential of bilayers was systematically studied. It was established that for a bilayer coverage exceeding 0.20, the net zeta potential became independent of the substrate and the supporting layer zeta potentials. Then, the asymptotic values of the zeta potential of the bilayer approach 1/√2=0.71 of the bulk zeta potential of the particles forming the external (second) layer. This behavior was interpreted theoretically in terms of the electrokinetic model derived previously for monolayers. It was also concluded that results obtained in this work can be exploited for interpretation of polyelectrolyte film formation in the layer by layer (LbL) processes and protein adsorption pertinent to the antigen/antibody interactions.