Optimization Of Detergent-Mediated Reconstitution Of Influenza A M2 Protein Into Proteoliposomes

We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process

Use of detergent in wool scouring

Wool scouring processes have been the subject of considerable analysis in recent years. However the necessary amount of detergent needed is still controlled by the personal judgement of experienced operators. No detergent measurement procedure, effective for commercial operation, is known. Optimal control of addition of detergent would naturally lead to reduced cost. This report is an attempt to analyse the detergent requirement of a wool scouring process with suggestions for further work

Evaluation of Ankom F58 Filter Bags Compared to Dacron Bags and Beakers for Analysis of Acid Detergent Fiber

Feed and fecal samples were analyzed to compare three methods of determining acid detergent fiber. Each sample was weighed into both Dacron and Ankom F58 fiber bags and then analyzed using an Ankom fiber analyzer. Results were then compared to the Van Soest beaker method. Ankom F58 bags helped reduce washout of small particles associated with Dacron bags, but fecal samples needed to be incubated in detergent for an extended amount of time to isolate acid detergent fiber material. Utilizing a technique that produces correct acid detergent fiber values is important for producers because these values are used as a proxy for calculating total digestible nutrients of feedstuffs

Synthesis of Rice Husk-Based Zeolit using Hydrothermal Method and Its Detergent Builder Properties

Detergents are cleaning agents that consist of a complex formulation such as surfactants, builders, bleaching, fillers and other additives. Detergent builder that commonly used is sodium tripolyphosphate (STPP) that are unfriendly environmentally. One of the detergent builders that is more environmentally friendly is zeolite. Therefore, in the present work, zeolite was synthesized using hydrothermal method by varying the temperature and characterised its detergent builder properties. Zeolite was synthesized by mixing sodium silicate (Na2SiO3) and sodium aluminate NaAl(OH)4 with the presence of NaOH. Firstly, a mixture of sodium silicate and sodium aluminate formed sol and gradually become gel. After that, the gel had been treated by hydothermal for 7 hours at different temperatures: 500C, 1000C and 1500C. Several characteristic techniques such as FTIR and XRD were applied to identify functional group and the crystal structure, respectively. Furthermore, the detergent builder properties were characterised using cation exchange capacity (CEC) and detergency test. The result showed that zeolite synthesized at 100 0C gave better washing capability than others

Synthesis of Rice Husk-Based Zeolit using Hydrothermal Method and Its Detergent Builder Properties

Detergents are cleaning agents that consist of a complex formulation such as surfactants, builders, bleaching, fillers and other additives. Detergent builder that commonly used is sodium tripolyphosphate (STPP) that are unfriendly environmentally. One of the detergent builders that is more environmentally friendly is zeolite. Therefore, in the present work, zeolite was synthesized using hydrothermal method by varying the temperature and characterised its detergent builder properties. Zeolite was synthesized by mixing sodium silicate (Na2SiO3) and sodium aluminate NaAl(OH)4 with the presence of NaOH. Firstly, a mixture of sodium silicate and sodium aluminate formed sol and gradually become gel. After that, the gel had been treated by hydothermal for 7 hours at different temperatures: 500C, 1000C and 1500C. Several characteristic techniques such as FTIR and XRD were applied to identify functional group and the crystal structure, respectively. Furthermore, the detergent builder properties were characterised using cation exchange capacity (CEC) and detergency test. The result showed that zeolite synthesized at 100 0C gave better washing capability than others

UJI TOKSISITAS BERBAGAI JENIS DAN KONSENTRASI DETERJEN TERHADAP TINGKAT KERUSAKAN INSANG DAN MORTALITAS IKAN MAS (Cyprinus carpio)

Toxicity is toxic relative character related with its potential to cause negative effect for living creature. Water pollution is the entrance of living creature, element, energy, and another component in water by human activity so that the water quality decreased into certain level which caused the water couldn’t function as the general function. The research purpose is to find out the influence of various kind and concentrate of detergent to gills corruptness and mortality of gold fish (Cyprinus carpio), to find out the detergent brand which has the highest level to gold fish gills corruptness and mortality, beside, the research aimed to find out the influence of combination of kind and concentrate of detergent to the corruption of fish gills and mortality of gold fish. The research was true experimental research. The experiment design used complete random design with 20 treatments, they were four kinds of detergents and each of detergent has 0.08 mg/L, 0.04 g/L, 0.07 g/L, 0.10 g/L, 0.13 g/L concentrates. While 4 detergents used were Attack, Rinso, Wings biru, dan Krim ekonomi. Sampling technique used to Simple Random Sampling. Research indicator was the level of fish gills corruption and mortality of gold fish.. Data analysis used two way ANAVA continued with Duncan’s Test 1%. According to two way ANAVA test, there showed that there is influence of detergent kind and concentrate to the level of gills corruptness and mortality of Gold fish (Cyprinus carpio). From the Duncan’s Test 1%, concentrate of Wings Biru brand 0.13 g/L had the highest influence to the level of gills corruptness and mortality of gold fish. Attack Detergent concentrate 0.008 had the smallest influence

Studies on methodology in dietary fibre analysis : a neutral detergent fibre method using glucoamylase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University

The dietary fibre content of foods is conveniently and rapidly determined by the neutral and acid detergent methods devised originally by Van Soest and associates. A serious disadvantage of the neutral detergent method relates to the interference caused by starch during filtration when the method is applied to cereals and cereal products. In these circumstances the results of neutral detergent fibre (NDF) measurements are variable and often over-estimated . A study of the starch-lipid reaction which takes place when cereal products are heated in Van Soest's neutral detergent solution showed that although the precipitate derived from pure wheat starch and lipid is soluble in hot water this action is often far from complete when much fibrous cereal matter is present. Much of the starch appears to be occluded in the NDF residue which then takes on a gummy-like character and tends to clog the filter. Southgate recently recommended purified amyloglucosidase from Aspergillus niger (Boehringer) for the purpose of hydrolysing starch in cereal samples before starting the neutral detergent extraction. Present studies have been concerned with the development of this enzymatic procedure with the aim of devising improved methodology and enhancing existing knowledge of the behavioural characteristics of amyloglucosidases from A. niger and from an alternative source, Rhizopus spp. Preliminary investigations showed that amyloglucosidase from A. niger (Boehringer) was completely effective as a starch hydrolysing agent in the pretreatment of a cereal substrate but that in order to use the enzyme economically it was necessary to use a semi micro version of Van Soest's neutral detergent extraction procedure. The main features of the new method are as follows: preparation of a subsample of lipid-free food sample of fine particle size; gelatinization of starch before enzyme treatment; treatment with the minimum quantity of enzyme (2 mg); extraction with neutral detergent at half the normal rate; separation of detergent solution from the residue by means of centrifugation; dehydration of the residue with acetone before filtration; special techniques for filtration, drying and weighing procedures. A table of NDF values for various cereal products determined by the semi micro procedure is presented. The results agree, for the most part, with the results of other workers in this field, the exceptions being for cornflakes, rolled oats and puffed wheat. The coefficients of variation for the NDF values compare favourably with those of other workers. A semi micro version of Van Soest's acid detergent method of evaluating dietary fibre was devised and is described with supporting analytical data. Tests performed with a low cost preparation of amyloglucosidase from Rhizopus spp (Sigma) showed that the crude enzyme was capable of fully hydrolysing the starch component of cereal products before commencing the neutral detergent extraction procedure but that it also seriously reduced the NDF values. In order to establish the cause of the discrepancies two approaches were made: an attempt was made to analyse the products of enzymatic hydrolysis; and a study of the effect of enzyme concentration on the yield of neutral detergent fibre was undertaken. The former approach proved impracticable, the latter suggested that either impurities in the crude enzyme preparation were responsible or the amyloglucosidase itself was active towards one or more components of dietary fibre. In order to determine which of the alternative explanations was correct small amounts of the crude enzyme preparation were purified by means of anion exchange chromatography using DEAE cellulose and one of two buffer systems, one based on citrate-phosphate, the other on tris-HCl. The citrate-phosphate conditions reported by Pazur and Lineback et al for the column separation of amyloglucosidase of A. niger were found to be quite unsuitable for the enzyme from Rhizopus spp. and a new set of conditions had to be determined for this enzyme. The activity of small amounts of the purified enzyme (< 1mg) was estimated by an improvised visual method using buffered 1% wheat starch, and the effect of the enzyme on cereal fibre was determined by means of the semi micro neutral detergent procedure using 0.08-0.2 g wholemeal flour as a substrate. It was found that both crude and purified forms of the enzyme caused a loss of ca 30% NDF from wholemeal flour, from which it was concluded that amyloglucosidase from Rhizopus spp was not a suitable enzyme for use in the neutral detergent method of measuring fibre. A literature review of the known chemistry of the amyloglucosidases of A. niger and R. delemar showed that differences in molecular structure reported by Pazur and others could account for their different electrophoretic properties. In the light of the present work it appears that another important biochemical difference between these enzymes relates to the activity of the Rhizopus enzyme towards the dietary fibre component of cereals

Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by Loop-Mediated Isothermal Amplification (LAMP)

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology/Principal Findings:&lt;/b&gt; For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions/Significance:&lt;/b&gt; This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.&lt;/p&gt