Patch buffering

Chick embryo heart muscle cells were patterned and grown on a fibronectin (FN) surface patterned by PDMS stamping. The PBS solution (containing Ca2+ and K+) was used to stimulate spontaneous muscle contraction [198]. Laminar flows provide a reaction path (buffer plus 1-octanol) and a control patch (buffer only) for study of communication between excitable cells (cardiomyocytes) through gap junctions (see Figure 8.18) [198]. 

McCarron The experiments we did involved whole cell patch-clamp studies. We were aware of the pH changes that may occur with CCCP so we buffered protons with 30 mM HEPES rather than the usual 10 mM. 

Pull and fire polish patch pipettes when filled with buffer solution show a resistance of 3-5 MQ and a series resistance of 5-6 MQ. 

Other biomolecules that are of interest in p.CP are lipids and lipid bilayers. Supported lipid bilayers are very fragile assemblies that are formed by lipids that are organized into two opposing leaflets on hydrophilic surfaces, such as glass or mica substrates. These structures can be also patterned on solid substrates but the p.CP technique differs slightly from the ones that were applied for proteins or DNA. First, the bilayer has to be formed on the oxidized PDMS stamp from the buffer solution by lipid vesicle fusion. Second, printing has to be carried out in water, otherwise the bilayer will lose its structure.99 This method allows efficient and reliable transfer of membrane patches to glass surfaces. 

Fig. 5. Ultraviolet spectra of Cbz-Try with Af-bromosuccinimide in aqueous acetate buffer of pH 4,0. A. Recorded directly. B. Recorded as difference. From Patch-ornik et al. (1960).

With [Co(phen)3] + as oxidant for PCu(I), rate constants determined by the stopped-flow method approach zero at low pH, consistent with zero reactivity of the trigonally coordinated Cu(I) form. However, with [FelCNlel as oxidant there is sometimes difficulty in fitting rate constants to the relevant [H ] dependence, which leaves open the question as to whether the rates actually become zero (57). Whereas [Co(phen)3l + is believed to react with PCu(I) at both the remote (acidic) and the adjacent (hydrophobic) patches, [FefCNleP reacts predominantly at the latter. The instability of PCu(I) in solution at pH < 4.5 makes it difficult to settle this issue conclusively. The range of studies has been extended using the pH-jump method in which protein, at high pH (with relatively small concentration of buffer), is stopped-flow mixed with the redox reagent at low pH (with excess buffer), and this approach has been used more extensively in recent studies. 

This model was found to account well for the adsorption data of propranolol from a buffer or salt solution onto a Cis-bonded silica surface [84]. The results obtained, however, suggested that the surface was not homogeneous and would be better modeled by assuming that it consists in patches of two different t)q>es of sites (see later. Section S.2.4.2). 

Brown and Kaplan made use of the buffering property of oils in phenol solutions. Their formulation contained up to 95% phenol combined with oils. A patch test behind the ear had to be carried out before the facial peel. If there was skin necrosis, they reduced the strength of the phenol by gradually adding oil in small quantities until the right dose was found for the patient s skin. To increase the strength of the mixture, on the other hand, soap (saponified cresol) was added or the concentration of phenol was increased. 

Finally, we note that a growing body of evidence shows that the stability of a planar membrane can be enhanced by spreading it across a small aperture [97], For example, a DiPhyPC bilayer suspended across a 150nm radius orifice in a glass pipet remains intact when removed from buffer [150], This suggests that it may be possible to form arrays in which fluid, stable bilayer patches are surrounded by a patterned substrate that anchors the membrane. Air stability can also be achieved by coating a PSLB with a hydrophilic polymer film (e.g., a biospecifically adsorbed protein layer [23,149]). Both of these approaches maintain some degree of lateral lipid mobility in the membrane. 

Bhardwaj et al. [2] studied by in-situ real-time STM imaging the passivation of polycrystalline iron in borate buffer. They proceeded by alternating oxidation steps at increasing anodic potentials and reduction steps at cathodic potential. After reduction of the natural oxide at ftie cathodic potential, relatively flat surfaces were produced supposedly corresponding to the metal substrate. Upon oxidation at anodic potential, rougher surfaces were at first produced, with patches or clusters of nanometer dimensions. These patches were observed in the first image after the oxidation step. 

In an alternative, whole-cell configuration, the small patch of membrane is ruptured by suction or brief high voltage, resulting in continuity between the patch pipette solution and the cytosol. Diffusional equilibration occurs between the large volume of the pipette solution and the cytoplasm, so that within a few minutes the cytosolic ion composition is essentially that provided in the pipette solution, with the possible exception of ions for which the cell has a large buffering capacity, e.g. and Ca. In the whole cell... 

One of the key features of ABA enhancement of Ik,oui is that it persists even when cytosolic Ca" concentrations are buffered to low levels by the inclusion of EGTA in the patch pipette solution during whole cell reeording. This suggests that elevated [Ca ],., is not involved in the activation of Ik.oui t>y ABA. Indeed, when [Ca ], is experimentally elevated from approximately 2 nM to 200 nM in whole-cell patch clamp experiments, current through outward K channels is reduced [72], an effect which would oppose stomatal closure. At higher [Ca ],.y,s, outward current shows little Ca -sensitivity [23]. More recently, it has been shown that Ik oui sensitive to cytoplasmic pH. Its regulation by pH has consequences for ABA-induced closure, and will be discussed in Section... 

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