Centrifugation assay

Figure 9. Anti-PbTx antiserum inhibition of [ H]PbTx-3 binding to its receptor site in rat brain membrane preparations. Labeled toxin (0.5 nM in 1 ml PBS) was incubated with rat brain membranes (125 fig total protein) and increasing amounts of anti-PbTx antiserum (- -) or pre-immune serum (- -) for 1 hr at 4 C. Membrane-bound radioactivity was then measured in a centrifugation assay as previously described (8),...

Centrifugation assays were employed in all our studies to eireumvent the problem of [ H]MDA and [ HJMDMA absorbing onto glass filters. 

To illustrate the principles of an equilibrium dialysis experiment, we will describe the binding of [ H] acetylcholine to the nicotinic acetylcholine receptor (nAChR) in native membranes from Torpedo electro-plax. The data obtained from such an experiment are shown in Figure 10-7 where they are compared with data obtained from a centrifugation assay described below (Protocol 4.2). 

In the centrifugation assay the membranes settle with the ligand, which is bound to them. The free ligand remains in the supernatant. 

To appear in the pellet fraction after centrifugation, lamin must be assembled into relatively large aggregates (Lin and Fisher, 1990). Hence, the apparently abrupt transition in lamin behavior seen by microcentrifugation when mitotic/ meiotic lamin is mixed with interphase lamin at a ratio of 1 8 versus 1 4 (Fig. 3) does not, in our estimation, reflect an abrupt change in lamin polymerization. Rather, it reflects an operational peculiarity of the centrifugation assay. Centrifugation is nevertheless useful in that it is easily and rapidly performed. 

Several semiquantitafive adhesion assays, including flow chamber assays, spinning-disc assays, and centrifugation assays, apply controlled shear stress to adherent cells. 

Chu, L., Tempehnan, L.A., Miller, C. et al.. Centrifugation assay of IgE-mediated rat basophihc leukemia cell adhesion to antigen-coated polyacrihmide gels, AIChE J. 40,692,1994. 

In the procedure for the surface test (313), the vims is grown in a monolayer of baby hamster kidney cells and incubated in Eagles medium supplemented with tryptose phosphate broth and calf semm. After separation of the vims from the cells by sonification and centrifugation, amounts of the suspension containing 3 x 10 plaque-forrning units are dried on coversHps. The inoculated coversHps are placed in 5 ml of the disinfectant for 1, 5, or 10 min, then rinsed, sonicated, and assayed. 

Assay 90-110% of label claim (pick 20 tablets at random grind and mix weigh an amount of powder corresponding to five tablets dissolve compound sample and centrifuge excipients run a HPLC analysis on an aliquot of the supernatant), and... 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

Since both of these compounds are very large they cannot enter the pores, thus the pores are still free to hold small molecules. When the coated charcoal is added to the assay reagent mixture, the free (unbound) material is adsorbed while the larger bound fraction is not. Final separation is accomplished by centrifugation and decantation of the supernatant. 

White blood cells, red blood cells and cultured fibroblasts are commonly used to measure enzyme activities, especially for the diagnosis of inherited enzyme abnormalities. Leukocytes may be collected by sedimentation in viscous media such as Fycol. The collection of red cells presents no problem following centrifugation of anticoagulated blood. The assay of enzymes and fibroblasts requires appropriate tissue culture facilities and extensive experience in dealing with cultured human cells. 

Yellow fevert Aqueous homogenate of chick embryos infected with attenuated yellow fever virus 170 1 Centrifugation to remove cell debris 2 Freeze drying Infectivity-titration in cell cultures by plaque assay Tests to exclude extraneous viruses... 

Membranes (50 pi in a total assay volume of 100 pi) were incubated with UDP-Gal (0.1 mM) and MgSO (10 mM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 min. Reactions were stopped by heating at 100°C for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70°C for 5 min and centrifuged (13000g 5 min). Supernatants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70 C and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 

ChSS was fractionated on a column (550 x 15 mm) of DEAE Sepharo e Fast Flow using a Hiload System (Pharmacia), which was initially equilibrated in 0.005 M NaAc-bufFer pH 5.0. The sample was dissolved in water, the insoluble residue was removed by centrifugation and the supernatant was applied onto the column. After applying the gradient shown in Figure 1, the residual polysaccharides were washed from the column using 0.5 M NaOH. Fractions (23 ml) were collected and assayed by automated methods [2,3] for total neutral sugars and uronic acids. 

To start the recycling system assay, 1200 L of water and 600 mL of the technical enzyme (0.5 mL enzyme/L of water) were added to the 2500 L holding tank. A periodical addition of water and enzyme at the same concentration was necessary to compensate for losses caused by the peel residues and centrifugation. The open system assay was performed in the normal way as reference, without recycling or enzyme addition. 

The culture broth was recovered after 72 h of fermentation, the biomass removed and the total protein content measured. Broth aliquots with a protein content of 1 mg were collected and their pH regulated at different values ranging from 3.5 to 8.0. To each broth fraction, 50 mg of the microspheres sample, previously equilibrated at the corresponding pH, was added and the suspension left under stirring overnight. Then, the microspheres were removed by centrifugation and the protein content and the PG activity were assayed on the resulting supernatant. 

FIGURE 8. The reduction of fMet(O)-Leu-Phe by a human neutrophil and purified E. coli Met(O) peptide reductase. Each assay contained in a final volume of 30 /j1 25 mM Tris-HCl (pH 7.4), lOmM MgCl, 15 mm dithiothreitol 540pmole fMet(O)-[ H]Phe-Leu and Met(0)-peptide reductase. After incubating at 37 °C for 60 min, the incubation mixture is acidified and extracted with ethyl acetate. After centrifugation, an aliquot of the organic phase is removed and assayed for radioactivity. Reproduced by permission of Academic Press from Fliss and coworkers ... 

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

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