Cellulose filter assay

Cellulose filter assay Cell migration occurs through a porous filter... 

This assay is a variation of the cellulose filter assay that uses multiple arrangements of chemoattractant above and below the filter to distinguish chemokinesis and chemotaxis. In addition, the depth to which the cells penetrate the filter is determined. These features allow calculation of population parameters x, the random motility coefficient and X, a chemotaxis coefficient. 

Like the cellulose filter assay, cells migrate through a filter that separates two chambers, with the cells in the top chamber and chemoattractant in the bottom. Because polycarbonate filters are very thin, migration in two dimensions along the top surface of the filter is primarily measured. This assay does not distinguish between chemokinesis and chemotaxis. 

The modified cellulose filter binding assay is based on the tight binding of proteins to this kind of filter material. When a protein-nucleic acid mixture is filtered, proteins are retained on the filter while nucleic acids are washed through. However, nucleic acids are also retained on the filter if they are bound to proteins. Thus, free and protein-bound nucleic acids can be separated [43]. 

Cells are placed in a chamber above a cellulose filter and chemoattractant is placed below the filter. The number of cells that move through the filter is quantified. This assay measures migration in three dimensions, but does not distinguish chemokinesis and chemotaxis. 

Filter paper activity, which describes the overall cellulolytic activity of an enzyme preparation, was determined by the method of Mandels et al. (16). A 1 x 6 cm strip of Whatman no.l filter paper (Hillsboro, OR), which equals 50 mg of cellulose, served as the substrate and was added to the sample solution containing 0.5 mL of appropriate diluted enzyme (supernatant of culture broth) and 1.0 mL of 0.05 M citrate buffer (pH 4.8). After 60 min of incubation at 50°C, the hydrolysis was terminated by the addition of 3 mL of DNS solution, and the mixture was further assayed for reducing sugar content by the DNS method. One international filter paper unit (FPU) was defined as the amount of enzyme that releases 1 pmol of glucose/min under the assay conditions. Activities were reported as FPU/milliliter. 

Fig. 6. Time courses of reduction of celluloses by NaBH4 at pH 8.0,22°C. Unreacted reducing ends were determined by BCA assay. The celluloses are as in Table 4. FP, filter paper.

The purified proteins were assayed for enzyme activity on filter paper, swollen cellulose, BMCC, and CMC following the procedures stated by Irwin et al (5). 

Two types of filters are available polycarbonate and cellulose nitrate. Polycarbonate filters are used for endothelial chemotaxis assays. These filters are sided, with a matt and a shiny surface. The cells are allowed to adhere to the shiny surface prior to migration, and then stimulated to migrate to the matt surface. 

We will perform the Leaf disk test, also known as leaf disk assay or leaf disk choice test, the second of two bioassays of tannins in the diet of insects in this book. In this often used bioassay, leaf sections of a standard size are treated with the compound(s) in question. Several circular leaf sections ( leaf disks ) (Ali et al. 1999, Filho and Mazzafera 2000, Shields et al. 2008, Wheeler and Isman 2000) or cellulose membrane filters (Hollister and Mullin 1999, Larocque et al. 1999) are presented to a caterpillar in a choice experiment. We measure how much chow the caterpillar has consumed and whether any feeding inhibition is concentration dependent. Regardless of what compounds are being tested, leaf disk tests serve as an important tool in bioassaying feeding inhibitors and stimulants in insects. The cited references are examples of such studies. (In the first tannic acid experiment - Chap. 18 - the tannic acid was mixed into diet in varying concentrations.)... 

For immunoassay techniques, cellulose acetate filters offer no advantage over glass fiber filter paper, and the slow flow rate and the handling problems of cellulose acetate when compared with glass fiber make it less suitable for use when large numbers of samples are involved. The retention characteristics of GF/B filter paper are adequate for most immunoassay techniques. However, in receptor assays, the particle sizes are close to the minimum retention size of the GF/B filter paper, and cellulose acetate filters are being used for filtration of these samples. 

The preparation of the cyclic AMP binding protein from bovine muscle has been described [150]. It involves homogenisation, centrifugation, pH 4.8 precipitation and ammonium sulphate precipitation, followed by fractionation on DEAE cellulose. The preparation binds 0.3 pmol of cyclic AMP per ixg of protein and has an enzymatic activity of 24 pmol of P per jxg of protein per 10 min. Over 200 /ig of the protein can be quantitatively adsorbed on a single Millipore filter. The yield of binding protein from 500 to 1000 g of muscle is sufficient for more than 100000 assay tubes. The binding activity is stable for 18 months at -20°C. 

Cellulase is a complex of enzymes showing various types of activities. Cellulose substrates include highly resistant crystalline forms such as cotton, various types of microcrystalline cellulose such as Avicel and hydrocellulose, sulfite pulps such as Solka Floe, as well as filter paper and cotton fabrics. More susceptible substrates include swollen or reprecipitated cellulose, cellophane, and ball-milled cellulose. Most susceptible are the soluble derivatives (of low D.S.) such as carboxymethylcellulose and cellulose sulfate. It is not surprising that there are many assay methods to detect or measure cellulase (9). These methods differ markedly in sensitivity, and in cellulase components detected, depending on the substrate used, the effect measured, and the duration and conditions of... 

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