Tris buffer and

Fig. 10.4.2 The effects of temperature (left panel) and pH (right panel) on the peak intensities of the Balanoglossus luminescence reaction. In the measurements of the temperature effect, 0.5 ml of 0.176 mM H2O2 was injected into a mixture of 1.2 ml of 0.5 M Tris buffer (pH 8.2), 0.3 ml of luciferase, and 1 ml of luciferin, at various temperatures. For the pH effect, the Tris buffer (pH 8.2) was replaced with the Tris buffers and phosphate buffers that have various pH values, and the measurements were made at room temperature. From Dure and Cormier, 1963, with permission from the American Society for Biochemistry and Molecular Biology.

The method is more sensitive than the biuret method and has an analytical range from 10 ju,g to 1.0 mg of protein. Using the method outlined below this is equivalent to sample concentrations of between 20 mg l-1 and 2.0 g l-1. The relationship between absorbance and protein concentration deviates from a straight line and a calibration curve is necessary. The method is also subject to interference from simple ions, such as potassium and magnesium, as well as by various organic compounds, such as Tris buffer and EDTA (ethylenediamine-tetraacetic acid). Phenolic compounds present in the sample will also react and this may be of particular significance in the analysis of plant extracts. 

The histone octamer of nucleosome core particles was cross-linked by dimethylsuberimidate and isolated from the DNA by precipitation in 3 M NaCl (0.05 M sodium phosphate buffer, pH 7.0). The cross-linked octamer, dissolved at low ionic strength, was reconstituted by mixing with DNA at 1.0 M NaCl (pH 8.0 Tris buffer) and dialyzed against 0.6 M NaCl in the same buffer. The reconstituted particle had properties similar to those of the cross-linked core particle. It sedi-... 

The separating or the resolving gel mix is first prepared by adding water. Acrylamide, Tris buffer and SDS according to the table mentioned below. 

Fig. 3. (a) Reaction of pyridoxal 5 -phosphate (PLP) with an amino-terminal amino group of hemoglobin (Hb). The reagent is in the form of a Schiffs base with tris(hydroxymethyl)aminomethane [77-86-1] (Tris) buffer, and the reaction is a transamination, (b) The resulting unstable Scbiff s base is reduced with... 

Sephadex G-50 has been prepared for your use. The dry beads have been preswollen in water for several hours, fine particles that may slow column flow have been removed, and the gel has been equilibrated in Tris buffer. If chromatography columns are not available, one can easily be constructed from a glass tube (2.5 X 40 cm), cork stoppers, Tygon tubing, a screw clamp, and small glass tubing as shown in Figure E4.4. With the screw clamp closed, add 10 to 15 mL of Tris buffer and insert a small piece of... 

Again, transfer 3.0 mL of the pH 7.5, ethidium bromide-Tris buffer solution into the cuvette. Add 15 /xL of the unknown DNA solution. Mix well and place in the spectrofluorimeter. Record the fluorescence intensity, Z7 [. The measured fluorescence is due to both DNA and RNA (if present). The amount of fluorescence due to ethidium-RNA can be eliminated by digesting the RNA with ribonuclease. To the cuvette containing pH 7.5, ethidium bromide-Tris buffer and unknown DNA, add 2 fiL of ribonuclease solution. Mix well and incubate the cuvette at 37°C for 20 minutes in a water bath. After incubation, measure the fluorescence intensity, (Atotal). Compare this with the original measurement taken on the unknown DNA (Atota ). Is RNA present in your DNA sample Calculate the concentration of DNA in your unknown solution as described in the Analysis of Results. The possibility exists that the solution of ribonuclease may contain a fluorescent impurity. What control experiment should be done to correct this situation ... 

Suspend the pellet in 15 mL of standard Tris buffer and transfer to a 50-mL Erlenmeyer flask. 

Figure 8.5 Pomegranate by-product (PBP) consumption by E° mice attenuates atherosclerotic lesion development, in association with reduction in macrophage oxidative stress and Ox-LDL uptake. E° mice consumed PBP (17 or 51.5 mg gallic acid equivalents/kilogram/day) for 3 months. Control mice received only water (placebo). At the end of the study, the mice aortas as well as the mice peritoneal macrophages were harvested. (A) Atherosclerotic lesion size determination. (B) Total macrophage peroxide levels were determined by the DCFH-DH assay. (C) For determination of macrophage paraoxonase 2 (PON2) lactonase activity, cells (2 x 10e) were incubated with 1 mmol/L dihydrocoumarin in Tris buffer, and the hydrolysis rate was determined after 10 min of incubation at 25°C. (D) The extent of Ox-LDL (25 pg of protein/ milliliter, labeled with FITC) uptake by the mice macrophages (1 x 10e) was determined by flow cytometry. Results are expressed as mean S.D. of three different determinations. = p < 0.01 versus placebo.

A DNA extraction protocol that has proved useful for most ancient tissues is a modification of the protocol initially published by Blin and Stafford.20 Approximately 0.1 g of small pieces of soft tissue is added to 5 ml of extraction buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 10 mg/ml dithiothreitol (DTT), and 0.5 mg/ml proteinase K. Incubation at 37° with gentle agitation overnight will allow most or all of the tissue to go into solution. An equal volume of phenol, equilibrated with 1 M Tris-HCl (pH 8.0), is added. When the phenol is being equilibrated, care should be taken to use uncontaminated Tris buffer and to measure the pH only on aliquots that are removed from the water phase and then discarded. Two phenol extractions and one chloroform extraction are performed, and the water phase is concentrated and purified on a Centricon 30 microconcentrator (Amicon, Danvers, MA). The reten-tate can be stored frozen, preferably in a few aliquots. In all cases solutions should be manipulated with DNA-free positive displacement pipettes. 

CopB was shown to catalyze ATP-driven copper(I) and sUver(I) transport into native membrane vesicles of En. hirae. Since only inside-out oriented ATPase molecules were active in this transport assay, this corresponds to copper extrusion by CopB in vivo. Copper transport by vesicles took place only under reducing conditions. Cu(I) rather than Cu(II) was thus the transported species. Use of null mutants in copA, copB, orcoj Aand copB> made it possible to attribute the observed transport to the activity of the CopB ATPase. Copper transport exhibited an apparent for Cu+ of 1 pM and a Umax of 0.07 nmol/min/mg of membrane protein. " Ag+ was transported with a similar affinity and at a similar rate (Solioz and Oder-matt, 1995). Since Cu" " and Ag+ were complexed to Tris buffer and dithiothreitol present in the assay, the values must be considered as relative only. The results obtained with membrane vesicles were further supported by evidence of " Ag+ extrusion from whole cells, preloaded with this isotope. Again, transport depended on the presence of functional... 

Prepare the upper reservoir buffer by dissolving 6 g Tris buffer and 28.8 g glycine in sufficient distilled water to yield a final volume of 1 liter. Check the pH of the solution. If it is not 8.3, adjust the pH with a small amount of Tris or glycine. 

Prepare solution D (upper gel buffer) by dissolving 8.9 g Tris buffer and 40.0 ml l.OM H3PO4 in sufficient distilled water to yield a final volume of 250 ml. The final pH of the solution should be 6.5 to 6.7. 

Figure 3 is a plot of the pmn at 25°C calculated by Equation 10b as a function of buffer molality m (m = madd = sait) Data obtained in the sulfate-free seawater I are connected by dashed lines, and those for seawater II (with sulfate) are joined by a solid line. Two features of these results require fmther study—the separation of the two curves for tris buffers and the separation and curvature of the lines for bis-tris. It seems unlikely that the presence of sulfate can alter the ion-ion interactions sufficiently to cause departures from Equation 9 of the magnitude (0.02 in pmn at m = 0.02) found with tris buffers. Consequently, specific interactions, possibly complexation with cations, are suspected. In this case, the differences in calculated pmn would be real. As will be seen presently, measurements of cells without liquid junction suggest that this is the case see Table V). 

Rinse grids first in Tris buffer and then in 2 baths of PBS (30 sec each) and dip in water. 

Ketkar M, Green V, Schnieder P, et al. 1979. Investigations on the carcinogenic burden by air pollution in man Intratracheal instillation studies with benzo[a]pyrene in a mixture of Tris buffer and saline in Syrian golden hamsters. Cancer Lett 6 279-284. 

Desorption kinetics provide additional information that contributes to our understanding of surface phenomena such as the specific effects of tris and bicarbonate buffers on monolayers. Stable condensed mono-layers were expanded on tris buffers, and ionization appeared enhanced (8). Unstable, expanded monolayers did not expand further on tris buffers, but K data (Table I) showed a consistent decrease in the apparent pKa when fatty acids were spread on tris buffers. 

Ox liver mitochondria were swollen in hypotonic Tris buffer and then were shrunk in hypertonic sucrose. This technique liberated a mitochondrial glycoprotein which was purified by preparative electrophoresis... 

Figure 10. Inhibitory effects of peptides from chymotryptic hydrolysates of modified bovine serum albumin (BSA) on hydrolysis of BSA by bovine a-chy-motrypsin. BSA was acetylated and then reductively methylated. Eight ml (0.87 mg/ml) of this product in 0.012M Tris buffer and 0.012M CaCl2, pH 7.7, was incubated at 37°C for 24 hr with 200 fig of a-chymotrypsin. The solution was then boiled for 10 min. Aliquots of this mixture of peptides were then added in the indicated proportions of BSA to the peptides. The various ratios of BSA to peptides in assay were 17 1 (%) 3.5 1 (M) 1.7 1 ( ). BSA alone (O). The BSA concentration was 0.2 mg/ml in 0.02M borate buffer at pH 8.2 ana the a-chymotrypsin was 3.2 fig/ml. The hydrolysis was followed according to a modification of the procedure by Lin et al. (62) [Figure from Ref. 56].

Turn on the recording spectrophotometer and the UV lamp Allow the mstm-ment to warm up for about 15 minutes. Set the wavelength to 260 nm. During the warm-up period, prepare samples by transferring 1 or 3 mL of Tris buffer into a quartz cuvette and an equal volume of purified a-lactalbumin solution into a matched cell. Place the cuvette with buffer only m the sample beam and adjust the absorbance to zero. Remove the blank cuvette and replace with the cuvette containing a-lactalbumin Read and record the absorbance at 260 nm If the is greater than 1 0, dilute the sample with a known volume of Tris buffer and repeat the reading... 

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