Profiling the expression of mitogen-induced T-cell proteins by using multi-membrane dot-blotting

High throughput technologies are standard methods for analysis of the proteome. Multi-layer multi-well plate dot-blotting system (MLDot) technology is a high-throughput dot blotting system that provides a simple, cost-effective approach for protein expression profiling in multiple samples. In contrast to traditional dot blot, MLDot uses a layered stack of thin, sieve-like membranes in place of a single nitrocellulose membrane. Therefore, up to 10 membranes can be prepared from the samples arrayed in a single 96-well plate. We describe the ability of MLDot to detect the predicted changes in protein expression following multiple mitogen treatment of T-cells. We compare the levels of the phopshorylated forms of CREB, Jun, and Akt in Jurkat T-cells as detected by MLDot to those measured by a gel-based assay. We also describe the ability of MLDot to detect differences in the levels of phosphorylated Akt in Jurkat cells as compared to primary lymphocytes.     

Panning of a phage VH library using nitrocellulose membranes: application to selection of a human VH library

We have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies.     

Identification of glycoproteins on nitrocellulose membranes and gels

In this article we describe a procedure for the detection of glycoproteins on gels employing the periodic acid-Schiff’s reagent. In addition, a number of staining protocols and direct binding ELISA, employing antibodies and lectins, are described for the identification and quantitation of glycoproteins after their immobilization by dot, slot, or Western blotting onto nitrocellulose membranes. We document, in detail, the conditions (i.e., the effect of solvent and detergents) for the immobilization of one specific family of O-linked glycoproteins, namely mucins. However, taking into account our suggestions, these procedures should be applicable to other types of glycoprotein.     

Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule

Molecular profiling of human biopsies and surgical specimens is frequently complicated by their inherent biological heterogeneity and by the need to conserve tissue for clinical diagnosis. We have developed a set of novel 'tissue print' and 'print-phoresis' technologies to facilitate tissue and tumor-marker profiling under these circumstances. Tissue printing transfers cells and extracellular matrix components from a tissue surface onto nitrocellulose membranes, generating a two-dimensional anatomical image on which molecular markers can be visualized by specific protein and RNA- and DNA-detection techniques. Print-phoresis is a complementary new electrophoresis method in which thin strips from the print are subjected to polyacrylamide gel electrophoresis, providing a straightforward interface between the tissue-print image and gel-based proteomic techniques. Here we have utilized these technologies to identify and characterize markers of tumor invasion of the prostate capsule, an event generally not apparent to the naked eye that may result in tumor at the surgical margins ('positive margins'). We have also shown that tissue-print technologies can provide a general platform for the generation of marker maps that can be superimposed directly onto histopathological and radiological images, permitting molecular identification and classification of individual malignant lesions.     

Determining proteome-wide expression levels using reverse protein arrays in fission yeast

Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast-tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.     

Solution of the problem of nonspecific binding in solid-phase noninstrumental dot immunoassay

In this work, we evaluated blocking properties of various reagents used in dot immunoassay test systems on nitrocellulose membranes employing carbon-based diagnosticum to determine antibodies to tetanus anatoxin, thermostable toxin of Yersinia pseudotuberculosis, and HIV-I. The results support the usage of a nonionic detergent Tween 20 which efficiently inhibited nonspecific protein binding to the solid-phase reagent surface and did not decrease sensitivity of the analysis.     

Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen-antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.     

Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots

We have determined that sodium salicylate, a water-soluble fluor which we use routinely for fluorography with polyacrylamide gels, is also useful for fluorography with thin-layer media. Detection of 3H-labeled material applied to thin-layer chromatography plates, or nitrocellulose membranes, can be enhanced up to 150-fold after treatment with an aqueous solution of 2 M sodium salicylate, while detection of 35S-labeled material is enhanced only about 2-fold. We demonstrate the utility of sodium salicylate fluorography in detecting 3H-labeled palmitic acid following thin-layer chromatography and 3H-labeled proteins following blotting to nitrocellulose.     

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.     

Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting

A selection of different membranes commonly used to bind proteins in blotting and dot binding assays were investigated for a range of properties which would influence their performance. Large differences were observed in the membranes' ability to bind increasing amounts of protein, the effect of incubation times on the quantity of protein bound and the loss of proteins from the membranes following their incubation with different detergents or protein blocking agents. These differences could only partially explain the observed performance of the membranes when used as protein adsorbants in immunoassays and when different buffers were used for the electro-transfer of several different proteins to a range of membranes.     

Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca²⁺ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.     

A novel probe Au(III) for chemiluminescent image detection of protein blots on nitrocellulose membranes

A new method, based on the chloroauric acid-enhanced luminol chemiluminescence, is established for the chemiluminescent imaging detection of protein blots on nitrocellulose membranes. After transferring to the nitrocellulose (NC) membranes, various proteins in human serum can be easily detected using this method. Simplicity and wide applicability are achieved, without the need of expensive antibodies or tedious immunoassay procedures. Furthermore, neither noxious materials nor radioactive pollution is produced. The successful detection of proteins is due to the binding of Au(III) to the protein blots and the chemiluminescent character of the enhanced luminol signal. As a novel chemiluminescent detection method, it offers significant biological analytical potentials in biochemistry and in molecular biology.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

CCA paper: a new two-dimensional cyanuric chloride-activated matrix for universal application in molecular biology

A novel two-dimensional cyanuric chloride-activated (CCA) paper has been developed. It is composed of a cellulosic base, covalently bound cyanuric chloride, and microprecipitated complex cyanuric chloride-sodium chloride crystals on its surface. CCA paper covalently binds nucleic acids and proteins. Its binding capacity for nucleic acids is about 400 micrograms/cm2. Sealed into nitrogen-filled bags and stored at -20 degrees C, it retains its binding activity for at least a year and is always ready for use. CCA paper has been successfully used for capillary and electroblotting of DNA, RNA, and proteins (Southern, Northern, and Western blotting) as well as for dot tests. Furthermore, it was applied to colony and plaque hybridization. A unique property of it is that it permits the staining of proteins after blotting and subsequent performance of radioimmunological detection of specific protein components. This has proven advantageous in two-dimensional Western blotting experiments. Of further importance is its ability to bind DNA fragments from one up to several hundred bp from polyacrylamide sequencing gels.     

A dot-blot assay for the low density lipoprotein receptor

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein.     

Chromogranins, widespread in endocrine and nervous tissue, bind Ca2+

The proteinaceous components of the secretory vesicle contents isolated from bovine adrenal medulla bind Ca2+ (number of binding sites, 152 +/- 52 nmol Ca2+ per mg protein; dissociation constant, 54 +/- 8 microM (n = 5)). SDS-polyacrylamide gel electrophoresis and 45Ca2+ binding of the proteins following their separation and blotting on nitrocellulose revealed that Ca2+ binds to chromogranins. Moreover, it was shown that the chromogranins, like other known Ca2+-binding proteins, can be specifically stained with a cationic carbocyanine dye. The Ca2+-binding function of the chromogranins described here, in conjunction with recent findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution of chromogranins in many different endocrine and nerve cells, points to the general importance of these proteins in the metabolism of Ca2+.     

Glycosylation of the human interferon-gamma receptor. N-linked carbohydrates contribute to structural heterogeneity and are required for ligand binding

The contribution of N-linked carbohydrates to human interferon-gamma receptor (hIFN-gamma-R) structure and function was investigated in four tumor cell lines of various tissue origin. Western and ligand blotting of native and deglycosylated, affinity-purified hIFN-gamma-R of the monocytic cell line U937 and the lymphoid cell line Raji revealed that the different sizes of hIFN-gamma-R from U937 (103 kDa) and Raji (90 kDa) cells are reduced upon either metabolic inhibition or enzymatic deglycosylation of N-linked carbohydrates to a common size of the receptor molecule with an apparent molecular mass of 73 kDa for both cell lines, indicating that heterogeneity in hIFN-gamma-R size is largely due to differential glycosylation. In all cell lines investigated, inhibition of N-linked glycosylation or modulation of carbohydrate processing did not prevent receptor transport to the cell membrane, but blocked hIFN-gamma binding capacity of membrane-expressed receptor molecules, as revealed by specific binding of hIFN-gamma-R-specific monoclonal antibody and specific binding of 125I-labeled hIFN-gamma. These data suggest that a lack of complex-type N-linked carbohydrates is associated with a complete loss of receptor function, i.e. high affinity binding capacity. Recovery of hIFN-gamma binding of deglycosylated receptors was achieved upon affinity purification and adsorption to nitrocellulose membranes, indicating that the carbohydrate side chains themselves do not directly contribute to the ligand binding epitope but seem to be essential for appropriate conformation of the receptor protein in the cell membrane.     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.