Phosphates assays

The initial mixture and each time point are then assayed for doxorubicin and lipid. Lipid concentrations can be quantified by the phosphate assay (see above) or by liquid scintillation counting of an appropriate radiolabel. Doxorubicin is quantified by an absorbance assay (see below). The percent uptake at any time point (e.g., t = 30 minutes) is determined by %-uptake = [(D/L), =30minutes] x 100/[(D/L) inuiai]. Doxorubicin can be assayed by both a fluorescence assay and an absorbance assay, but we find the latter to be more accurate. The standard curve consists of four to five cuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL samples to be assayed are of the same volume. To each tube is added 0.9 mL of 1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems such as DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to 15 seconds, until the detergent turns cloudy. Samples are allowed to cool, and absorbance is read at 480 nm on a UV/Visible spectrophotometer. 

The initial mixture and each time point are then assayed for ciprofloxacin and lipid. Lipid can be quantified using the phosphate assay (64,65) or by liquid scintillation counting. Ciprofloxacin is quantified by an absorbance assay following removal of drug from lipid by a Bligh-Dyer extraction procedure (78) (see below). The percent uptake is determined as described in the section Remote Loading of Doxorubicin into DSPC/Cholesterol (55 45) Large Unilamellar Vesicle. ... 

Day 2 Precipitation of Pi, Cation Exchange with Dowex 50, and Initial Glucose-1-Phosphate Assays... 

Collect the filtrate and record its volume. Remove duplicate 0.05, 0.1, 0.2, and 0.5 ml samples of the filtered solution for the inorganic phosphate and 7-min phosphate assays described in steps 5 through 9. Place these duplicate samples in small, labeled glass test tubes. 

If the phosphate assay reveals that an excess of inorganic phosphate is still present in the incubation filtrate (i.e., if an intense blue color forms in the unhydrolyzed 0.1 ml sample), add 1 g ofMg(OAc)2 4 H20, adjust to pH 8.5 with 14% ammonia, cool the solution, and filter it again. Then repeat the phosphate assays. (Step 10 is not usually necessary.)... 

Figure 13-3 Quantitative phosphate assay to quantify phopholipids.

Thin-layer chromatography Cholesterol assay Phosphate assay... 

Day 2 Thin-Layer Chromatography of Lipids, Quantitative Cholesterol Assay, Folin-Ciocalteau Assay, and Preparation for Phosphate Assay... 

Flgnre 2-16. Absorbances observed with increasing amounts of inorganic phosphate assayed by a modification of the Fiske-Sub-barow procedure. 

An enzyme sequence electrode for phosphate assay based on AP and GOD has been devised by Guilbault and Nanjo (1975b). Glucose-6-phosphate (G6P) was used as the substrate for AP ... 

Barclay RL. ATP (Adenosine Tri-Phosphate) Assay An Innovative Method for Measuring Biomass Levels in Pulp and Paper Mills, 1994 Papermakers Conference Proceedings. Norcross Tappi Press, 1994. 

Lipid analysis is typically performed either by incorporating a nonexchangeable radiolabel marker, such as (14C) or (3H) cholesteryl hexadecyl ether (CHE), into the vesicle membrane, or by analyzing the phosphate content and extrapolating the result according to the original composition of the vesicles. Both approaches assume that the label concentration and vesicle composition do not change on vesicle preparation or subsequent manipulation. The phosphate assay is carried out as follows ... 

D2. Davies, B. M. A., Gordon, A. H., and Mussett, M. V., A mouse urine phosphate assay for parathyroid hormone with certain applications. J. Physiol. (London) 180, 79-95 (1955). 

The Pj liberated by acid or enzymatic hydrolysis of the inositol phosphates may also be assayed. An anion-exchange HPLC column, which separates the inositol phosphates, is coupled to a second column that contains immobilized alkaline phosphatase. The Pj that is released by the enzyme is measured color-imetrically. The system can detect 1 nmol of inositol phosphates in a single sample to indicate levels of IP3 of 13 to 40 nmol/g of tissue. The sensitivity of the phosphate assay is increased to the picomolar level with malachite green as a reagent (Dean and Beaven, 1989 Palmer and Wakelam, 1989). 

FIGURE 13.8 General scheme for a microsphere kinase/phosphates assay. The fluorescence of the conjugated polyelectrolyte is quenched as the quencher-labeled peptide (black line with a filled black circle) is brought in dose vicinity to the microsphere, due to interaction between Ga and P03. ... 

Numerous bioassays format have been designed using derivatives of the mono-functionalized europium cryptate such as 47 (commercially available from CISbio, see http //www.htrf.com). Since the last reviews [127] some new applications Eu " C [BP.BP.BP] cryptate were developed through the HTRF technology, as caspase assay [128], telomerase assay [129], leukotriene b4 assay [130], inositol-1-phosphate assay [131], minisequencing [132], and MutS-DNA interaction [133]. Recently, the HTRF technology has been used in the study of protein-protein interactions and the authors demonstrated that measured values compare favorably with those calculated from independent experiments [134]. 

Two factors must be taken into account to obtain quantitative data. It is necessary to use a sufficient delay between rf pulses to ensure that all nuclei are fully relaxed before the next rf pulse is applied (relaxation agents may be added), and the effect of NOE must be eliminated. Consequently, C s-NMR is normally not readily quantifiable. Precision of phosphate assays by NMR is consistently within 0.2-0.6%, comparable to results obtained using chromatographic methods. 

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