Pipes buffer

Prepare a QD solution in 25 mM PIPES buffer, pH 7.0 (reaction buffer), at a concentration of lOOpg/ml. 

Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES) buffer Dissolve 1.385 g PIPES (Sigma) and 0.235 g sodium acetate (Merck) in 20 ml demineralized water. 

Reaction buffer Mix 20 ml PIPES buffer with 50 ml NaCl solution and add another 30 ml demineralized water in a 250-ml flask. The pH is adjusted to 6.5 with 0.1 M HC1. Fill to the 250-ml mark with demineralized water. 

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). 

Figure 12. Titration of GdCl with Ca2 -ATPase (24). The solutions contained 0.05M TMA-Pipes buffer, pH 7.0, 24.4 pM GdCl, (A) or 49pM GdCl, (B), and the noted concentrations of Ca2 -ATPase. The value of t obtained by extrapolation of the solid line to the infinite protein concentration is the average of the enhancements at Gd3 sites 1 and 2, denoted by c , and iBi, respectively, in the text.

The possibility of using the electron paramagnetic resonance properties of Gd3+ to probe its environment in and interactions with biological molecules has previously received little attention in the literature (40). However, the possibility exists that Gd2+ will be a sensitive EPR probe for characterizing macromolecular biological systems such as the Ca2+-ATPase. The EPR spectra of Gd3+, which has S = 7/2. in neutral water and in two different buffers are shown in Figure 13A. The linewidths were found to be independent of pH over the usable range of these buffers and independent of temperature between 4 and 30°C, The spectrum of Gd2+ in neutral water is centered around 3248 G, with a linewidth of 530 G. As shown, Gd3+ in Pipes buffer, but not in Tes buffer, yielded a spectrum similar to that of the aqueous Gd2+ solution. On this basis, all of our Gd3+ EPR and NMR studies... 

The primary fixative was a mixture of 3% glutaraldehyde, 1% formaldehyde freshly prepared from paraformaldehyde, and 0.75% tannic acid in 0.05 M piperazine-N,N -bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0. As tannic acid may lower the pH of the fixative, it is recommended that this is checked before use and adjusted to 7.0 with 1N NaOH if necessary. The addition of low-molecular weight tannic acid to the primary fixative consistently improves the preservation of membranes and microtubules.2... 

After primary fixation the algal and root samples were rinsed in 0.1 M PIPES buffer, pH 7.0, then twice in 0.1 M sodium cacodylate buffer, pH 6.8, and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 6.8, overnight at 4°C. The osmium solution prepared for the root samples was added with 0.7% potassium ferricyanide in order to improve osmium penetration in the root tissues. This was particularly necessary for the 2 h control roots and roots from RO-treated seed, due to their relatively low water content. The replacement of PIPES buffer with cacodylate buffer before osmication was necessary, as PIPES reacts with osmium, producing a dark precipitate. 

All reactions were run for 40 minutes with the following specific reagent concentrations being used DNA, 15 pg mL dithiothreitol (DTT), 2.0 mM PIPES buffer, 5 mM, pH 7.0, 23 °C. This figure corresponds to one that originally appeared in reference 319 and has been reproduced with permission... 

Lipoplexes were prepared in a Hepes/mes/pipes buffer at pH = 8.0, at different charge ratios from liposomes obtained with compounds 2 and 4 described above, in association with the co-lipid DOPE (cationic lipid/DOPE 1 1), and were compared to those obtained from stable cationic lipids 1 and 3. 

The non-ionic detergents Triton X-100 and NP40 are frequently used in studies of the cytoskeleton. They are applied either prior to fixation with a cross-linker in the presence or absence of precipitant fixatives or after fixation to remove remaining lipids. Generally, 0.3% of detergent in PIPES buffer containing 0.1 mM EGTA is used for 1 to 3 min. 

PIPES buffer base piperazin-N,N -bis(2-ethanesulfonate). TRIS buffer. 

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