Proteins interference from

The reagent sequence is specific for endosulfan and phosphamidon. Other insecticides, e.g. organochlorine insecticides, such as endrin, aldrin, dieldrin, DDT and BHC, organophosphorus insecticides, such as malathion, parathion, dimethoate, quinalphos, phorate and fenitrothion, or carbamate insecticides, such as baygon, car-baryl and carbofuran do not react. Neither is there interference from amino acids, peptides or proteins which might be extracted from the biological material together with the pesticides. 

If the fluorometric procedure is used, with protein precipitation, then bilirubin will not interfere with the hexokinase procedure. Even if protein precipitation is not resorted to, the interference from bilirubin does not become significant until one is dealing with an infant in severe jaundice. This can be seen in Table III. 

Modify a purified bait protein by adding an aliquot of crosslinker to achieve a molar excess of 2-10 moles per mole of protein (protect from light). For NHS ester-containing reagents, react in 0.1 M sodium phosphate buffer at pH 7.2-7A. Avoid using any other amine-containing buffer components, such as Tris or imidazole, which will interfere with the reaction. 

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PILL tube (20 cm X 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4+ and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36],... 

Various protein binding techniques are reported (32-42) for the determination of calcitriol in plasma. Generally, a preliminary purification step is required to avoid interference from other plasma components. 

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. 

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

Another way to detect small molecules in the final formulated protein product without the interference from the protein signals is to remove the protein by ultrafiltration. Figure 12.4 compares a section of the proton NMR spectra of a biopharmaceutical protein product before (upper spectrum) and after (bottom spectrum) the protein was removed by ultrafiltering the sample with a Centricon-10 (Millipore Corp, Bedford, MA). Removing protein results in a flatter baseline (bottom spectrum). If small molecules are present in a protein sample, the removal of the protein may allow for unobstructed detection of the small molecules. In this case, a small amount of acetate ( 1 pg/rnl) is detected in the sample [bottom trace, Figure 12.4], Figure 12.5 shows that spikes of 10 p.g/ml of acetate and MES into the protein sample are fully recovered after the ultrafiltration to remove the protein. This example demonstrates that the interference of protein with the detection and quantitation of small-molecule impurities in a formulated protein product can be effectively eliminated by ultrafiltration. 

The CE-SDS method is a size-based separation technique generally applicable to proteins from 10 to -200—300kDa. The specificity is generally tested against the formulation buffer and any other possible contaminant proteins. There is usually no interference from the formulation buffer with the assay. For samples that contain contaminant proteins with a hydrodynamic size of 10—200kDa, the method is not specific. 

The results obtained with ISEs have been compared several times with those of other methods. When the determination of calcium using the Orion SS-20 analyser was tested, it was found that the results in heparinized whole blood and serum were sufficiently precise and subject to negligible interference from K and Mg ([82]), but that it is necessary to correct for the sodium error, as the ionic strength is adjusted with a sodium salt [82], and that a systematic error appears in the presence of colloids and cells due to complexa-tion and variations in the liquid-junction potential [76]. Determination of sodium and potassium with ISEs is comparable with flame photometric estimation [39, 113, 116] or is even more precise [165], but the values obtained with ISEs in serum are somewhat higher than those from flame photometry and most others methods [3, 25, 27, 113, 116]. This phenomenon is called pseudohyponatremia. It is caused by the fact that the samples are not diluted in ISE measurement, whereas in other methods dilution occurs before and during the measurement. On dilution, part of the water in serum is replaced by lipids and partially soluble serum proteins in samples with abnormally increased level of lipids and/or proteins. 

Colorimetric assays are commonly used in molecular biology and biotechnology laboratories for determining protein concentrations because the procedures and their instrumentation requirements are simple. Two forms of assays are used. The first involves reactions between the protein and a suitable chemical to yield a colored, fluorescent, or chemiluminescence product. Second, a colored dye is bound to the protein and the absorbance shift is observed. Disadvantages of both these methods include limited sensitivity at below 1 pg/mL, interferences from buffers, and unstable chromophores (Jain et al. 1992). 

Although the spectrophotometric assay of proteins is fast, relatively sensitive, and requires only a small sample size, it is still only an estimate of protein concentration. It has certain advantages over the colorimetric assays in that most buffers and ammonium sulfate do not interfere and the procedure is nondestructive to protein samples. The spectrophotometric assay is particularly suited to the rapid measurement of protein elution from a chromatography column, where only protein concentration changes are required. 

Fluorescent labels are advantageous because they can be used not only for sequential detection methods on the same blot with minimal potential interference, but also for detection prior to protein extraction from the membrane. For example, after visualization of proteins with a fluorescent label, the blot can be photographed and specific bands marked with a pencil, either directly on the membrane or through a plastic bag. The latter method leaves a permanent indentation on the membrane. The blot can then be probed with antisera (i.e., immunoblotted unitbja). 

When colours are added to a food system, their characterisation is often more difficult due to interferences from other materials in the food or difficulty with their extraction from the food. This is particularly the case for high-protein foods, which bind colours very tightly and can make their quantitative analysis very difficult. However, analysis for azo-dyes in soft drinks is generally straightforward using modern methods. There is less interference than in other food systems and as the colours are already in solution, and not bound to other materials, this makes the analysis easier. In some cases, the colours can be analysed without prior concentration and in others they have to be concentrated by solid-phase extraction methods, for example, Ci8 cartridges followed by elution with a small volume of methanolic ammonia. 

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