Protein transfer from isoelectric focusing gels: The native blot

The nonelectrophoretic transfer of proteins from thin (0.5 mm) isoelectric focusing gels to nitrocellulose was complete in 1 h. Blotting was bidirectional with 60% of the protein transferred to the top blot and the remaining 40% to the bottom blot. The use of nondenaturing transfer buffers permits proteins to be blotted in the native state. Immunological determinants are preserved and enzyme activity can be detected on the blots.     

A New Spectrophotometric Method for the Detection of Lipase Activity Using 2,4-Dinitrophenyl Butyrate as a Substrate

A rapid and sensitive assay for the detection of lipase activity is described. The method is based upon the increase in absorbance at 360 nm due to the formation of the 2,4-dinitrophenolate anion during the enzymatic hydrolysis of 2,4-dinitrophenyl butyrate. The substrate is used in an emulsified form. Using a diode array spectrophotometer with internal referencing a correction can be made for absorbance changes due to clearance of the emulsion during hydrolysis. The small reaction volume and the high extinction coefficient of the product makes the method applicable for detection of both low substrate and low enzyme concentration.

Four lipases were tested: lipase from porcine pancreas, Candida cylindracea, Pseudomonas sp. and Aspergillus niger. All enzymes are readily able to catalyse the hydrolysis of 2,4-dinitrophenyl butyrate.     

组织型纤溶酶原激活剂的发光分析法及其应用

 本文建立了组织型纤溶酶原激活剂(t-PA)活力的发光固相测定方法。用氨基乙基丁基异鲁米诺(ABEI)标记纤维蛋白原(Fg),在一定条件下,t-PA作用于固相(包被ABEI-Fg),产生纤维蛋白的降解产物。测定可溶性降解产物的发光强度,即能计算t-PA活性。该方法的标准曲线范围对t-PA为0.156IU/mL～40IU/mL。灵敏度可达0.156IU/mL。回收率为98.6％(n=27)。批内批间变异系数分别为6.6％及10.3％。该方法曾用于检测细胞培养液中提取t-PA样品及t-PA基因表达时培养液中t-PA的活性。也曾用于检测从组织中纯化t-PA样品及血浆中t-PA活性的测定。文中讨论了该方法与其它方法优缺点的比较。     

Comparison Between Different Assays for Superoxide Dismutase-Like Activity

The direct and indirect methods for assaying the superoxide dismutase activity of a compound are compared. With the use of a direct method. the mechanism of the catalysis of O₂-dismutation by the tested compound can be determined. while with the indirect method it cannot. and this may lead to misinterpretation of the results. Assuming that the catalysis occurs via the 'ping-pong' mechanism, both the direct and indirect methods are limited to the determination of values of k_cat ≥ 10⁵M^-1s^-1 and k_cat ≥ 3 × 10⁶M^-1s^-1. respectively. Moreover, many side reactions may occur with the indirect method which may interfere with the measurements. Nevertheless. the indirect method approximates better the in vivo conditions than the direct method, and a tested compound that has high SOD activity using a direct method and low SOD activity using an indirect method. will most probably be a poor SOD mimic in vivo.     

A new simplified assay for larval migration inhibition

, , , and 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22: 1183–1185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-intestinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.