Hepes, solution preparation stock

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

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. 

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. 

HEPES) was obtained from Sigma. NH Cl, CaCl2, MgCl2, LiCl, NaCl, KCl, ethanol and methanol were purchased from Fisher. DMSO and quinine sulfate were procured from Aldrich. The water used was purified by MILLI-Q water system (Millipore). Stock solutions (2 mM) of the 4-methylcoumaro-cryptands were prepared in DMSO and diluted with water or methanol. Stock solutions (0.1 M) were prepared in 50 50 ethanol-water for 4-methylcoumaro-crown ethers and diluted with the same solvent mixture. 

Artificial cerebrospinal fluid (aCSF) 3 mM KCl, 150 mM NaCl, 2 mM CaCli, 5 mAf HEPES. Prepare using stock solutions 1, 3, and 4, and adjust pH to 7.4 with l.OA/NaOH (stock solution 5). 

A solution of 1 mg.mr NBD-PE (Avanti) In HEPES buffer (10 mM HEPES, 35 mM sodium nitrate, pH 7.4) was prepared In vesicle form. 60 ul of this was added to 100 ul of stock AChR solution and sonicated for different periods of time, yielding a final system of vesicles approximately 2 molt NBD-PE In I Ipid component, assuming a mean molecular weight of 750 gjK>l for the soybean lecithin. 

Extract buffer lx XB salts (lOOmM KCl, 0.1 mM CaCl, 1 mM MgCl from 20x XB salts stock solution), 50mM sucrose (1.5M stock filter-sterilize and store in aliquots at -20°C), lOmM HEPES (IM stock, titrated with KOH so that pH is 7.7 when diluted to 15mM, should require about 5.5ml of ION KOH for lOOmL, dilution drastically changes the pH of HEPES, so pH must be monitored after dilution filter-sterilize, and store in aliquots at -20°C). Prepare about lOOmL. 

For the 4 mM amino acid stock, first, individual stocks are prepared. Prepare a 20 mM stock solution of tyrosine and 100 mM stock solutions for the other 19 amino acids. L-tryp-tophan must be dissolved in 100 mM Hepes, pH 8.0. L-aspar-tic acid, L-cysteine, L-glutamic acid, and L-methionine must be dissolved in 100 mM Hepes, pH 7.4. All other amino acids are dissolved in MilliQ water. Sonication or heating to 60°C could improve solubility. However, some stocks (in particular W, D, E, N, C, Y) won t dissolve completely and have to be handled as suspensions. For the 4 mM amino acid mixture, take 2 mL of each the 100 mM amino acid stocks and 10 mL of the 20 mM L-tyrosine stock and make up to 50 mL with MilliQ water. For the 8 mM amino acid stock, weigh in all compounds and dissolve the powders in MilliQ water. The stock remains turbid. 

Atomic Absorption Spectroscopy was conducted for the half-apo GpdQ enzyme to confirm the presence of one equivalent of iron. Standards containing 20,40,60, 80, and 100 ppb Fe, Zn, Mn, and Co were prepared in 50 mM buffer (HEPES, pH 7) from analytical stock solutions. Protein samples were prepared using the same buffer. 

The gap between the glass shde and covershp is filled with aqueous solution, in which the sample may be dissolved or immersed. Since single molecule fluorescence imaging is a kind of extremely low-level light detection, contamination should be as minimized as possible. Hence, the aqueous solutions must be prepared with freshly-purified ultra pure water. The aqueous stock solutions such as the pH buffers and sahnes can be frozen until use in order to eliminate any increase in bacteria. The additives to the aqueous solutions can also influence the background of microscope field. As a pH buffer, we often use phosphate, HEPES, PIPES, citrate, and acetate. 

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