Tris-buffered saline , preparation

Tris-buffered saline with Tween-20 (TBS-T) prepare lOX stock with 1.37 M NaCl, 0.2 M Tris-HCl, pH 7.6, 1% Tween-20. 

Prepare IX Tris-buffered saline Tween20 (TEST) buffer per manufacturer s directions. Fill water reservoir with deionized water. Empty waste container if appropriate. 

Standard (or wash) buffer phosphate-buffered saline (PBS, 10 mM sodium phosphate, pH 7.4, 150 mM NaCl) or Tris-buffered saline (TBS, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4), and 0.5 M EDTA, pH 8.0 prepare fresh as required. 

The pH of a tris buffer solution prepared as described above is a function of temperature and salinity and can be estimated from the following equation Almgren et oL, 1975) ... 

An aliquot of proteins prepared from 3T3-L1 adipocytes was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/Tween 20 (TEST) (10 mM Tris-HCl, pH8.0,150 mM NaCl,... 

In contrast to tissues, tissue culture cells are readily lysed with detergent [11]. Adherent cells from a culture plate are first scraped using a rubber policeman into a small volume of phosphate-buffered saline (137 mMNaCl, 2.7 mM KC1, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.3) and harvested by centrifugation at 1500 x g for 10 minutes at 4°C. The cellular pellet is resuspended in ice-cold TE so that 1 mL contains 100 million cells. After the addition of 10 volumes of freshly prepared digestion buffer (10 mM Tris-Cl, pH 8, 0.05 EDTA, pH 8, 0.5% Sarcosyl, and 100 pg/mL proteinase K), the sample is incubated at 50°C for 3 hours. DNA is recovered by ethanol precipitation after extraction with phenol, phenol-chloroform, and chloroform as described earlier. 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

A 10 mmol L stock coelenterazine solution was prepared by dissolving coelenterazine (Nanolight Technology, Prolume Ltd. Pinetop, AZ, USA) in methanol for use at a final concentration of 10 /tmol L". All coelenterazine solutions were stored at -20 °C and working solutions were kept on ice in the dark during preparation. Diluent buffers comprised distilled water (dH20), Phosphate buffered saline (PBS), Buffer A (10 mmol L Tris [pH 7.8], 1 mmol L EDTA, 0.6 mol L NaCl), 7H9 medium supplemented with Tween-80 with or without 10% OADC (oleic acid, albumin, dextrose, catalase), Luria-Bertani (LB) broth with or... 

The mixture is brought to pH 8.0 with 2 M Tris-HCl, and chromatographed on an acid-sepharose 4B column equilibrated with borate-saline buffer (see preparation of RT) at 4°C. 

Sample preparation. Although there is not a gold standard for sample preparation and target extraction for analysis by immunoassay, most use saline buffers (e.g., phosphate-buffered saline, Tris saline buffer, high-salt buffers) at neutral pH to prevent interference and inhibition of antigen-antibody binding. Similarly, there is no standardized extraction method for DNA-based assays, and extraction efficiency is matrix dependent. However, unlike immunoassays, DNA-based assays will withstand the use of harsh conditions. 

Fig. 1. Separation of pyBHK EF-2 from the cellular ADP-ribosyltransferase by immunoabsorbant chromatography. A 50 jug sample of an EF-2 preparation containing the cellular transferase activity was applied to a 250 jul column of Sepharose 4B resin coupled to Pseudomonas toxin A antibody. Unbound sample was washed from the resin with phosphate-buffered saline containing 1 vaM DTT (PBS). Protein bound to the immunoabsorbant was dissociated and eluted in 1 M propionic add containing 1 mAf DTT. Acid-containing fractions were immediately neutralized with 2 M Tris-Base. A portion of each fraction was assayed for EF-2 ( — ) by the transfer of [ C]-adenosine from NAD to the EF-2 in the presence of diphtheria toxin fragment A [6]. A second portion of each fraction was assayed for pyBHK ADP-ribosyltransferase activity (o—o) by the transfer of [ C]-adenosine from NAD to EF-2 in our standard cellular transferase reaction mixture [7] which was supplemented with 4 jug of a pyBHK EF-2 preparation which lacked its own endogenous transferase activity. Acid precipitable radioactivity was detected using a liquid scintillation counter...

Potassium dihydrogen citrate 0.05 molal solution (pH=3.776 at 25 °C) is used for calibration purposes because it exhibits better stability than primary pH reference buffer solutions of tartrate or phthalate [44, 45]. The saline sodium citrate buffer (SSC) prepared from tri-sodium citrate and sodium chloride (pH=7.0) is applied in biochemistry. Citric buffers with different HjCit Na3Cit ratios are clinically effective, for example in reducing gastric acidity [46-48]. Compositions of buffers and corresponding pH values are presented in Table 3.7. 

For plasminogen-deficient fibrinogen from blood plasma, the anticoagulated blood was centrifuged and the plasma was frozen and washed with saline solution. Treated with charcoal and freeze-thawed. Dialysed versus Tris/NaCl buffer. [Maxwell and Nikel Biochemical Preparations 12 16 1968]. 

Oligonucleotide solutions are prepared in TE buffer pH 8 (0.1 M Tris-HCl buffer solution, ImM in EDTA). Aliquots are prepared and maintained at -20°C. Working solutions were conserved at 4°C. Hybridisation takes place in a 2 x SSC (saline sodium citrate, 30 mM sodium citrate buffer with 300 mM sodium chloride and pH 7.0) buffer containing 50% of formamide. 

P 82] Dilution-type mixing was accomplished with the fluorescent dyes acridine orange (0.01% solution in 20 mM in TE buffer see below) or trypan blue (prepared in 0.85% saline) contacted with buffer solution (TE buffer 10 mmol f4 Tris-HCl, pH 7.4, 1 mmol 1 1 EDTA, pH 8.0) [164]. Images were taken by a laser scanning confocal microscope. Profiling data analysis was employed along detection lines. 

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