Hepes, solution preparation

To minimize errors occurring when weighing the compounds, HEPES-buffered HBSS may be prepared from three stock solutions. Prepare 1L lOx stock no. 1... 

Preparation of PLL-j-PEG-Coated Metal Oxide Substrates. For polymer deposition on oxide substrates, a given PLL(x)- [y]-PEG(z) was dissolved in 10 mM HEPES [Sigma-Aldrich Inc., St. Louis, MO] at a concentration of 1.0 mg/mL. Unless otherwise noted, all HEPES solutions in this study were adjusted to pH 7.4 with 1.0 M NaOH. 

Silicon (100) wafers, passivated with silica, were employed as substrates. Prior to immobilization of PLL- -PEG onto the oxide surface, the wafers (0.5 cm 0.5 cm) were prepared by sonication in toluene (2 min) and in 2-propanol (10 min), extensively rinsed with ultrapure water (EM SCIENCE, Gibb-stown, NJ), dried under a gaseous nitrogen flow, and exposed for 2 min to an oxygen plasma (PDC-32G. Harrick Scientific Corp., Ossining, NY). The oxidized substrates were immediately transferred to a 1.0 mg/mL solution of PLL- -PEG in a 10 mM HEPES buffer and incubated there for 40 min. The polymer-coated substrates were then stored in a HEPES solution (in the absence of PLL- -PEG) until use in AFM experiments. Prior to AFM measurements, the polymer-coated substrates were withdrawn from solution, rinsed with a HEPES buffer and ultrapure water to remove free PLL- -PEG, and then dried under a nitrogen flow. 

Silicon and microsphere surfaces were prepared for measurement and/or polymer adsorption through a series of steps. These included ultra-sonication in toluene for 1 minute, ultra-sonication in iso-propanol for 10 minutes, rinsing with HPLC grade water, and exposure to an oxygen plasma (see above) for 2 minutes. Following this procedure, samples and/or tips were immediately transferred to a lOmM HEPES solution for storage or polymer adsorption. The polymer was adsorbed onto surfaces prepared in this fashion by immersion for 30 minutes in 10 mM HEPES solutions containing 1 mg/ml of PLL(20)-g[3.5]-PEG(2). 

These were prepared in a procedure similar to that of Scheme 1, except that the ferrocylacetic was replaced by ferrocenecarboxylic acid. All reactions were carried out in an ice bath. 80 mg of ferrocenecarboxylic acid were dissolved in 4 mL of a 0.15 M Na-HEPES solution, to form a slightly turbid pH 7.3 0.1 solution. When higher, the pH was lowered to this value by adding dropwise, with stirring, 0.1 M HCl.)... 

The SI70 supernatant (220 ml) was made to 40 % saturation with solid ammonium sulfate, stirred for 20 min, and then the precipitate was collected by centrifugation at 15,000 g for 15 min. The precipitate was suspended in small volume of buffer B-50 at pH 7.6 containing 20 mM HEPES/KOH, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, and 50 mM potassium acetate. The 60 % saturated ammonium sulfate solution was prepared similarly. Protein concentrations for 0 - 40 % and 40 - 60 % ammonium sulfate fractions were 4.2 mg/ml and 4.7 mg/ml, respectively. 

Using pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) as intraliposome pH indicator, the liposomes were prepared as above (as in section Preparation of 100 nm SSL Loaded with DOX via Transmembrane AS Gradient ) with the exception that pyranine (0.5 mM) was included in the hydration solution. Removal of untrapped pyranine was achieved by gel filtration on a Sephadex G-50 column, preequilibrated with either NaCl, KCl, sucrose or AS solution (according to need). All these solutions also contained lOmM Hepes buffer at the desired pH (usually pH 7.5). 

Dequalinium chloride (10 mM final) and paclitaxel (10 mM final) were dissolved in methanol in a round-bottom flask followed by removing the organic solvent with a rotary evaporator. After adding 5 mM HEPES, pH 7.4, the suspension was sonicated with a probe sonicator until a clear opaque solution of DQAsomes with encapsulated paclitaxel was obtained (usually for about one hour). To remove undissolved material, the preparation was centrifuged for 10 minutes at 3000 rpm. 

The solutions were prepared with different ionic strengths in different concentrations (10, 20, and 30 mM) in Tris-HCl and HEPES buffers and at pH 7, 8, and 9. 

Figure 27. Comparison of the L-Clu-induced integrated channel currents between two different preparations of GluR-incorporated BLMs upon injecting the identical concentration of l-GIu (0.10 nM). Applied potential +50 mV. Conditions 9.8 mM HEPES-NaOH (pH 7.6) containing 0.52 M NaCI, 0.19 mM CaCl2, 4.8 mM glycine, 24 pg mL of concanavalin A, 8.1 mM sucrose and 0.40 M formamide in both cis and trans side solutions. Proteoliposomes were injected only to the cis side and agonist solution was added to the trans side. Applied potential +50 mV. The l-GIu solution was injected to the trans side. ...

As for Basic Protocol 1, the LOX enzyme is prepared according to the Support Protocol. However, more care should be taken to reduce turbidity and other UV-absorbing materials caused by suspended lipid and pigments, especially when low activity is expected (requiring more enzyme addition). Triton X-100 is a UV absorber, and it causes more lipid and pigments to be suspended. Partial purification may be necessary. HEPES and PIPES buffers are not used in this method because of excessive absorption at 234 nm (0.5 to 0.6 absorbance for 0.1 M solutions) MES is useful with limitations (0.2 absorbance for 0.1 M solution). 

To resolve folded and unfolded conformations of the 387 nt Tetrahymena ribozyme, we use 8% acrylamide (29 1 mono bisacrylamide) in 34 mMTris, 66 mM Hepes (pH 7.5), 0.1 mM EDTA, and 3 mM MgCl2 (THEM3). Hepes is used instead of borate to maintain the native structure of the ribozyme (Buchmueller and Weeks, 2004 Pyle et ah, 1990), whereas the MgCl2 concentration is chosen to be just sufficient to maintain the RNA in its folded state. Twenty-five milliliters of acrylamide solution per gel is prepared and degassed using RNase-free water. 200 /iL 10% ammonium... 

Hepes is used at 10-25 mM and is added to medium from a stock solution (1 M) whose preparation is described in Appendix 1, Table A1.5. As well as Hepes, other zwitterionic buffers have been used in cell culture medium. TRICINE (lV-[Tris-hydroxymethyl)-methyl]gly-cine, pKa = 7.79 at 37°C) has been used in Eagle s MEM (Spendlove et al., 1971) and in Swim s 577 (Gardner, 1969) and TES, (iV-[(Tris-hydroxymethyl)methyl]-2-aminoethanesulphonic acid, pKa = 7.16 at 37°C) has been used in Eagle s MEM, BME and in medium 199 (Williamson and Cox, 1968 Massie et al., 1972). These buffers are used in varying concentrations (10-50 mM). Eagle (1971) suggests combinations of buffers which can be used to buffer the medium over the range 6.4-8.35. 

Acetylcholineesterase Bilayer lipid membranes were prepared by adding a solution of egg phosphatidylcholine and dipalmi-toyl phosphatidic acid dropwise into the surface of aqueous 0.1 M KC1/10 mM HEPES, near the Saran Wrap partition of a two compartment plexiglass cell. A portion of AChE solution in 10 mM Tris hydrochloride buffer solution of pH 7.4 was applied. The electrolyte level was momentarily dropped below the orifice and raised to form a membrane. The membranes were used as transducers for the reaction of AChE with ACh. An external voltage (25 mV) was applied across the membrane between two Ag/AgCl reference electrodes. Enzymatically generated hydronium ion causes transient current due to alteration of the electrostatic field by the ionization of dipalmitoyl phosphatidic acid. The response delay time was directly related to the substrate concentration where acetylcholine can be determined from 1 pM upto mM level. [113]... 

The ability of compounds 21, 22, 23, 25, and 28-32 to conduct cations was examined in planar bilayers composed of phosphatidylethanolamine (PE) painted across a 200 pm diameter aperture in a septum between two aqueous compartments filled with 600 mM KC1 (10 mM HEPES pH 7.2) (Figure 13). Bilayer quiescence was confirmed at 148 mV and bilayer stability was observed to be unaffected by the addition of up to 5 pL of DMSO. Stock solutions of the channel compounds (10 pM) were prepared immediately prior to use, and 0.1-5 pL (i.e. 1-50 pmol of substance) in DMSO was added to the stirred solution in the cis chamber. Typically, channel insertion into the bilayer occurred between 30 s and 30 min. 

The ability of E. coli PHB/polyP complexes to form calcium-selective channels in planar bilayers was investigated in the planar bilayer system described above (Figure 5). E. coli DH5a cells were made genetically competent to increase the concentration of PHB/polyP in the membranes. Then vesicles were prepared from the cell envelopes, and added to the cis side of a planar bilayer formed by synthetic 16 0, 18 1 PC between symmetric bathing solutions of 250 mM CaCl2, 5 mM MgCl2,10 mM Tris Hepes, pH 7.3. The complexes were allowed to insert spontaneously into the bilayer.27 No activity was observed in the absence of an applied... 

---