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At least 505 records · Page 28Linked to original sources
PubMed1988

Fibroblast growth factor effects on peripheral nerve regeneration in a silicone chamber model.

We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two-compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or growth factors that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic fibroblast growth factor (FGF) solution. FGF is a known angiogenic factor and a mitogen for endothelial cells, fibroblasts, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8-10 days. In vivo experiments, examined at 16 days post-implantation, revealed that spatial migration of all cellular elements (perineurial-like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF-treated ones. Most striking was the well-developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one-half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a well-developed structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.

Animals↗
PubMed2005

Solid phase microextraction ion mobility spectrometer interface for explosive and taggant detection.

Ion mobility spectrometry (IMS) is a rugged, inexpensive, sensitive, field portable technique for the detection of organic compounds. It is widely employed in ports of entry and by the military as a particle detector for explosives and drugs of abuse. Solid phase microextraction (SPME) is an effective extraction technique that has been successfully employed in the field for the pre-concentration of a variety of compounds. Many organic high explosives do not have a high enough vapor pressure for effective vapor sampling. However, these explosives and their commercial explosive mixtures have characteristic volatile components detectable in their headspace. In addition, taggants are added to explosives to aid in detection through headspace sampling. SPME can easily extract these compounds from the headspace for IMS vapor detection. An interface that couples SPME to IMS was constructed and evaluated for the detection of the following detection taggants: 2-nitrotoluene (2-NT), 4-nitrotoluene (4-NT), and 2,3-dimethyl-2,3-dinitrobutane (DMNB). The interface was also evaluated for the following common explosives: smokeless powder (nitrocellulose, NC), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2,4,6-trinitrotoluene (2,4,6-TNT), hexahydro-1,3,5-trinitro-s-triazine (RDX), and pentaerythritol tetranitrate (PETN). This is the first peer reviewed report of a SPME-IMS system that is shown to extract volatile constituent chemicals and detection taggants in explosives from a headspace for subsequent detection in a simple, rapid, sensitive, and inexpensive manner.

Butanes↗
PubMed2003

Protein microarrays using liquid phase fractionation of cell lysates.

We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.

Automation↗
PubMed2003

Identification of repertoires of surface antigens on leukemias using an antibody microarray.

We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria.

Antibodies↗
PubMed2003

Carbohydrate microarrays.

Sugar chains are abundantly expressed on the outer surfaces of the vast majority of viral, bacterial, protozoan and fungal pathogens, as well as on the membranes of mammalian cells. This class of carbohydrate molecule is without peer in structural diversity and is characteristically suitable for storing and displaying biological signals for molecular and cellular recognition. Exploring the biological information contained in sugar chains is an important topic of current postgenomic research. To facilitate these investigations, we have focused on the establishment of a carbohydrate-based microarray technology. Recently, we reported that a large panel of carbohydrate-containing macromolecules, including polysaccharides, natural glycoconjugates, and the mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a microglass slide to produce a large-scale carbohydrate microarray. In this review, we attempt to summarize our recent progress in using this technology to uncover the carbohydrate-based biological signals that are recognized by the human and animal immune systems. We also discuss the potential of various platforms of carbohydrate microarrays that were recently established and analyze the challenges to future development of carbohydrate microarray technologies and their applications.

Animals↗
PubMed2003

Microarrays to characterize protein interactions on a whole-proteome scale.

Protein microarrays contain a defined set of proteins spotted and analyzed at high density, and can be generally classified into two categories; protein profiling arrays and functional protein arrays. Functional protein arrays can be made up of any type of protein, and therefore have a diverse set of useful applications. Advantages of these arrays include low reagent consumption, rapid interpretation of results, and the ability to easily control experimental conditions. The ultimate form of a functional protein array consists of all of the proteins encoded by the genome of an organism; such an array would be the whole proteome equivalent of the whole genome DNA arrays that are now available. While proteome microarrays may not have reached the stage of maturity of DNA microarrays, recent developments have shown that many of the barriers holding back the technology can be overcome. Arrays of this type have already been used to rapidly screen large numbers of proteins simultaneously for biochemical activities, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. Eventually, functional protein arrays will be used to facilitate various steps in the drug discovery and early development processes that are currently bottlenecks in the drug development continuum.

Antibodies↗
PubMed2005

Peptide mass fingerprinting by matrix-assisted laser desorption ionization mass spectrometry of proteins detected by immunostaining on nitrocellulose.

We have developed an approach that allows peptide mass mapping by matrix-assisted laser desorption ionization-mass spectrometry of proteins visualized on a nitrocellulose membrane by immunochemical detection. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a nitrocellulose membrane and after blocking with a nonprotein-containing polymer such as polyvinylpyrrolidone 40 (PVP-40) or Tween 20, the proteins are stained with fount India ink. After incubation with primary and, if required, secondary peroxidase-coupled antibodies, immunochemically reactive proteins can be visualized using conventional enhanced chemiluminescence detection and assigned to the India ink-stained membrane by simple superposition. The proteins of interest are excised, submitted to "on-membrane" cleavage and the peptides are analyzed by mass spectrometry. Protein-based blocking reagents normally used in standard immunodetection protocols, such as skimmed milk, can be employed. We have obtained high-quality mass spectra of bovine serum albumin (BSA) detected on an immunoblot with an estimated amount of 100 fmol applied onto the gel, indicating the sensitivity of the present method. In addition, the approach is demonstrated with two other commercially available proteins, a serum protein, the successful identification of a tyrosine phosphorylated protein from total rat liver homogenate and serine phosphorylated proteins from an EcR 293 nuclear extract separated by two-dimensional (2-D) SDS-PAGE.

Animals↗
PubMed1995

Rapid screening of genetic polymorphisms using buccal cell DNA with detection by matrix-assisted laser desorption/ionization mass spectrometry.

A new approach is developed for the rapid and cost-effective detection of human genetic polymorphisms based on matrix-assisted laser description/ionization mass spectrometric (MALDI MS) detection using a nitrocellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as templates, followed by direct digestion with restriction enzymes and subsequent analysis by MALDI MS. The extraction of DNA from buccal cells provides a rapid and convenient means for sampling PCR-based diagnostic analysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatched primers and multiple primers. The MALDI MS methodology has been successfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detection of carbonic anhydrase and cystic fibrosis transmembrane conductance regulator as model genes. The detection of genetic polymorphisms following routine biological and clinical procedures with the MALDI MS method is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALDI MS method is several orders of magnitude faster than gel electrophoretic techniques. The method described herein should also be readily extended to other areas involving DNA screening and testing.

Base Sequence↗
PubMed2001

Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions.

Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha(2a) and anti-interferon-alpha(2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible).

Amino Acid Sequence↗
PubMed1986

The biology of human cytomegalovirus infection after bone marrow transplantation.

Human cytomegalovirus (HCMV) infection remains the most common infectious cause of morbidity after bone marrow transplantation (BMT). In a prospective study of 127 BMT recipients who received blood cultures for HCMV between days 28 to 105 after marrow grafting, HCMV viremia occurred in 68 patients (53.4%). Twenty patients (15.7%) had one or two positive cultures, and 48 (37.7%) had greater than or equal to three positive cultures. Fifty-nine patients (46.4%) had no viremia. HCMV-associated interstitial pneumonia (HCMV-IP) occurred in one-third of the viremic patients. Quantitative measurements of infectious HCMV or of HCMV DNA in lung tissue were made to determine whether HCMV replication correlated with clinical disease. Using DNA probes, viral DNA was measured by dot-blot hybridization, and this correlated with infectious HCMV. However, neither HCMV DNA nor HCMV viral titer correlated with time from the onset of pneumonia to death. The hypothesis is presented that HCMV-IP is caused by immunologic events induced after HCMV infection. In this model HCMV alterations in recipient cell surfaces induce donor alloreactivity to minor histocompatibility differences and lead to the subsequent pneumonitis which we term HCMV-IP. This model suggests that prevention of HCMV-IP will require early use of antiviral therapy or late use of immune response modification.

Animals↗
PubMed1992

A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.

A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.

Collodion↗
PubMed1993

A high temperature transfer procedure for detection of G protein gamma subunits by immunoblotting.

The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein gamma subunits in a suitable state for immunodetection. We describe a high temperature (70 degrees C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein gamma subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellulose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.

Amino Acid Sequence↗
PubMed1993

Silver-enhanced copper staining of protein blots.

Copper staining of protein blots has previously been shown to detect proteins rapidly, quantitatively, and sensitively. The sensitivity of this assay can now be enhanced nearly 10-fold with an additional step of silver staining. The silver-enhanced assay retains many of the virtues of copper staining, including a quantitative optical response with the amount of adsorbed protein, low variation between proteins, and reversibility. In addition, both copper staining and silver-enhanced copper staining do not detect nucleic acid, thus allowing for selective measurement of nucleic acid binding proteins or protein contamination in nucleic acid preparations. Silver-enhancement requires only a 5-min incubation with a single reagent. Since copper staining is performed first, it can be used to determine if the greater sensitivity provided by silver enhancement is desirable. For quantitation, an inexpensive reflectance scanning densitometer utilizing optical scanners and NIH Image is described.

Albumins↗
PubMed1993

A fast and sensitive method for detection of phospholipid-binding proteins on nitrocellulose membranes.

We describe a sensitive new method for specific detection and quantitation of phospholipid-binding proteins using two purified lipocortins as model proteins. The method consists of blotting proteins to nitrocellulose membranes followed by incubation with radiolabeled phospholipids and autoradiographic detection of bound phospholipids. It allows specific detection of phospholipid binding proteins to a threshold of about 100 ng/spot and their quantitation up to several micrograms as well as rapid analysis of specific phospholipid binding properties of individual proteins. If performed as a dot-blotting technique, the method permits the simultaneous analysis of a large number of samples and the establishment of Ca2+ and phospholipid binding curves. If blotting is preceded by electrophoresis, the method also allows specific detection and quantitative estimation of phospholipid binding proteins in crude biological preparations and may thus be useful for monitoring and analysis of these proteins in various biological samples.

Animals↗