Percoll reversibly inhibits superoxide dismutase

Incubation of pea leaf extracts (Pisum sativum L.) at 6 degrees C in isoosmotic media containing different Percoll concentrations significantly represses the total superoxide dismutase (SOD) activity in a concentration- and time-dependent manner. After 24 h incubation at 6 degrees C, 30-45% Percoll concentrations bring about an inhibition of Mn-SOD activity of more than 50%. Isozyme Cu,Zn-SOD II is affected to a lesser extent, with a maximum inhibition of 36% at high Percoll concentrations, whereas isozyme Cu,Zn-SOD I undergoes only slight variations. However, dilution of the samples followed by electrophoresis completely removes the Percoll inhibitory action. Results suggest that superoxide dismutases could be adsorbed onto the Percoll surface through electrostatic interactions.     

Visualization of antigenic proteins blotted onto nitrocellulose using the immuno-gold-staining (IGS) method

A new and simple method for the detection of antigenic proteins blotted onto nitrocellulose was developed. After transfer of spinach stromal proteins and purified phosphoribulokinase immunolabeling was performed with phosphoribulokinase antiserum, followed by a) Protein A-labeled colloidal gold particles, and b) by horseradish peroxidase conjugated Protein A and substrate mixture. The Protein A-Gold method is at least twofold more sensitive than the Protein A-peroxidase procedure. Incubation of immunolabeled nitrocellulose replicas with 0.1 M glycine, pH 2.2, removes the antibody-Protein A-Gold complexes quantitatively without influencing the antigenicity of the immobilized proteins. The replicas can be re-used for immunostaining with other antisera. The versatile applicability of the immuno-gold-staining method suggests that it is a true alternative to the peroxidase assay.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

A method for quantitating nanogram amounts of soluble protein using the principle of silver binding

A highly sensitive and quantitative assay for measuring protein in solution based on the capacity of protein to bind silver is described. In this procedure, protein samples are first treated with glutaraldehyde and then exposed to ammoniacal silver. After 10 min, the reaction is terminated by the addition of sodium thiosulfate and the optical density measured at 420 nm. The useful range of the assay for the majority of standard proteins tested lies between 15 and 2000 ng. This represents a 100-fold increase in sensitivity over the Coomassie brilliant blue dye-binding procedure. There is little or no interference from carbohydrates, nonionic detergents, or ethanol, and pretreatment of protein samples with Bio-Gel P-2 to remove salts, thiol agents, EDTA, and sodium dodecyl sulfate makes this procedure compatible with most commonly used buffers. The cost in terms of silver utilization is nominal with a typical assay involving 10 samples tested in triplicate amounting to less than $0.02 U. S.     

Percoll密度梯度离心法分离大鼠肺Ⅱ型上皮细胞

目的建立大鼠肺泡Ⅱ型上皮细胞（AT-Ⅱ）的改良体外原代培养法。方法采用循环冲洗、支气管灌洗、胰蛋白酶消化、滤网过滤并用轻比重及重比重PercolL密度梯度离心,分离AT-Ⅱ。原代培养后通过碱性磷酸酶（alkaline phosphatase,AKP）法和电镜鉴定AT-Ⅱ。结果经AKP染色和电镜鉴定所得的AT-Ⅱ纯度为（80．2±6．3）％,每只大鼠可分离出1×10^7AT-Ⅱ。结论采用Percoll密度梯度离心法能分离出较高纯度的AT-Ⅱ。     

A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.     

A high-throughput competitive scintillation proximity aminoacyl-tRNA synthetase charging assay to measure amino acid concentration

An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of scintillation proximity assay (SPA) technology to measure aminoacyl-tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive scintillation proximity assay (cSPA). This cSPA takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same nonradiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid. Under equilibrium conditions, in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration becomes irrelevant and the enzymatic isotopic dilution technique becomes the simple isotopic dilution technique. Due to the exquisite specificity of the reaction, a crude mixture of tRNAs and aaRSs can be used to detect the concentration of a particular amino acid without interference from noncognate amino acids. When used to monitor aminopeptidase M activity, this assay produced similar results in time course and inhibition experiments as compared with a traditional fluorescent assay. High-throughput compatibility was demonstrated by screening 12,000 compounds against aminopeptidase M in 384-well microtiter plates with Z factors ranging from 0.53 to 0.70. This competitive assay can be used as a general method to detect amino acids at concentrations less than 100 nM and to monitor enzyme activity in biological samples, and it is amenable to high-throughput screening.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.     

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.     

Refinement of the Coomassie brilliant blue G assay for quantitative protein determination

Minor modifications of the Bradford method [1976) Anal. Biochem. 72, 248-254) increase the sensitivity of the Coomassie brilliant blue G microassay for protein quantitation. This is reached by selecting the proper dye source, by alterations in the dye, ethanol, and phosphoric acid concentrations, and by including Triton X-100 in the reaction mixture. The modified assay conditions allow proteins to be detected above 0.2 micrograms and from a solution above 0.4 micrograms/ml. The extinction coefficient of the dye-protein complex was 0.144 +/- 0.010 for 1 microgram bovine serum albumin in the final reaction mixture of 1 ml. In general, the modified procedure exhibits the same specificity, advantages, and drawbacks as described for the original Bradford assay.     

Determination of bromide in canine plasma using ion chromatography

A new ion chromatographic procedure has been developed and validated for the determination of bromide in canine plasma. Following a simple dilution, samples were separated on a Metrosep A Supp 5 column. The mobile phase was an isocratic mixture of 2.2 mM Na(2)CO(3), 1.0 mM NaHCO(3), and 1% acetonitrile, with a flow-rate of 0.7 ml/min. The procedure produced a linear curve over the concentration range of 50-2500 microg/ml. The development of the assay permitted the determination of therapeutic levels after oral administration of potassium bromide to dogs being treated for epilepsy.     

Assay of protein in the presence of high concentrations of sulfhydryl compounds

A simple procedure is described for removal of Triton X-100 from protein samples by adsorption of the detergent on a commercial copolymer in bead form. The concentration of detergent can be lowered to approximately 0.01% with no loss or dilution of protein.     

A colorimetric enzyme assay using Escherichia coli to determine nutritionally available lysine in biological materials

A novel bacterial method is described for determining nutritionally available lysine in protein foods with a lysine auxotroph of Escherichia coli (strain M2626). Lysine-dependent synthesis of the induced enzyme bT-galactosidase is determined by a colorimetric method. With this approach sensitivity is increased ca 100-fold and assay time decreased to ca 2 h. The improved procedure was applied to the assay of lysine present as the free amino acid or in small peptides, and after enzymic pre-digestion in vitro with a mixture of pronase and intestinal peptidases, to pure proteins and a variety of feed meals and rice cultivars. In addition, heat treatment of complex samples was shown to lower their content of available lysine, as judged by the decreased nutritional response of the E.coli strain.     

Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

Use of a two-step Percoll gradient for separation of loggerhead sea turtle peripheral blood mononuclear cells

In order to determine a suitable procedure for isolating peripheral blood mononuclear cells (PBMCs) from loggerhead sea turtles (Caretta caretta), blood was collected using three different anticoagulants (sodium heparin, sodium citrate or potassium EDTA) and separated using a single step commercially-prepared arabinogalactan gradient of 1.077 g/ml density or multiple step Percoll gradients between 1.053 and 1.076 g/ml density (40-60% stock isotonic Percoll suspension). Heparinized blood centrifuged over a two-step 45/55% (1.059/1.070 g/ml) Percoll gradient yielded 99 to 100% mononuclear cells at the 45/55% interface. Mononuclear cell viability ranged from 85 to 97% with cell yields up to 9.2 x 10(6) cells/mL. An unexpected finding was a population of low density granulocytes migrating to 40% (1.053 g/ml) and 45% Percoll layers in the multiple step gradients. These granulocytes could be eliminated from the PBMC preparation by use of the two-step 45/55% Percoll gradient. Isolated PBMCs can be used for cellular immunology and toxicology studies on these threatened marine organisms for which other tissues can usually be obtained only sporadically from post-mortem specimens.     

Development and validation of a fast and sensitive chromatographic assay for all-trans-retinol and tocopherols in human serum and plasma using liquid-liquid extraction

A sensitive HPLC assay for all-trans-retinol, alpha-tocopherol, and gamma-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 microl aliquots of the supernatant (equivalent to 6.7 microl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C(18) S3 ODS2 column with a methanol-water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for alpha-tocopherol and 250 ng for gamma- and delta-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.     

Serum antibodies to individual cytomegalovirus structural polypeptides in renal transplant recipients during viral infection

In a longitudinal study we examined by immunoblotting (IB) the development and the evolution of the humoral immune response against individual cytomegalovirus (CMV) structural polypeptides in a total of 80 serum samples from 13 renal transplant recipients showing serological evidence of CMV infection and five renal transplant recipients with an anti-CMV antibody level unchanged over the observation period. The results showed that the IB reactivity at the time of transplantation may be a good index of the host's humoral immune status against CMV; by using this procedure it is possible to identify a seroconversion by the detection of antibodies reacting with some intermediate molecular weight proteins in sera examined at high dilution. Furthermore, IB is a very sensitive procedure also for IgM detection as it anticipates the positivity of the enzyme immune assay for IgM.     

A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.