Parameters affecting hybridization of nucleic acids blotted onto nylon or nitrocellulose membranes

Nine different nylon and nitrocellulose membranes were compared utilizing four different methods of attaching the nucleic acid target. Nylon membranes repeatedly demonstrated increased sensitivity as compared to nitrocellulose membranes. Sensitivity could also be enhanced by mildly denaturing the target prior to attachment onto the membrane. This was achieved by either UV cross-linking or baking.     

Dried gel hybridization in place of Southern hybridization for detection of Listeria monocytogenes DNA fragments

A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used.     

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.     

Purification and analysis of mitochondrial membrane proteins on nondenaturing gradient polyacrylamide gels

A nondenaturing gradient polyacrylamide gel electrophoresis method is described for the resolution of membrane proteins. Bovine heart inner mitochondrial membranes were solubilized in Triton X-100 and individual complexes were identified by staining for activity and protein. Succinate dehydrogenase was isolated by band excision and shown by electrophoresis under denaturing conditions to be highly purified. In addition, the electrophoretic transfer of NADH dehydrogenase to nitrocellulose was demonstrated. The enzyme was identified on the resulting blot by activity staining and the binding of monospecific antibodies.     

Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus

A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.     

Quantitative molecular hybridization on nylon membranes

A study of DNA hybridization to DNA covalently bound to nylon membranes was made in order to develop a quantitative method for molecular hybridization using a nylon-based matrix. Chloroplast DNA was covalently attached to nylon membranes by irradiation at 254 nm. Under hybridization conditions the initial rate of DNA loss from the nylon membranes was 5-10% per 24 h, while under comparable conditions DNA bound to nitrocellulose membranes was lost at a rate of 38 to 61% per 24 h. Several sets of hybridization conditions were examined to select one giving reasonable hybridization rates and minimal loss of bound DNA. Under the conditions selected [Denhardt's solution (D. Denhardt, 1966, Biochem. Biophys. Res. Commun. 23, 641-646), 0.5 M NaCl, 0.1% sodium dodecyl sulfate, and 31.4% formamide at 50 degrees C for 92 h], hybridization was observed to be 29% more efficient on nylon membranes than on nitrocellulose. Several attempts to remove previously hybridized DNA from nylon membranes proved only partially successful. Reuse of the membranes, therefore, was of limited value. Quantitative hybridization of total radiolabeled tobacco cellular DNA to cloned tobacco chloroplast DNA attached to nylon yielded results similar to those previously reported using nitrocellulose membranes. However, use of nylon membranes greatly facilitated the manipulations required in the procedure.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

Use of nitrocellulose membranes as a scaffold in cell culture

Nitrocellulose membranes, one of the most important and oldest cellulose derivatives, are commonly used for nucleic acid and protein detection in research and diagnostic applications. However, a limited number of studies have explored whether they can act as scaffolds for cell growth. In this study, we investigated this polymeric material for its ability to support the growth of human cells. Eight established cell lines were examined for adherence, growth, spread, and survival on nitrocellulose membranes by optical microscopy after hematoxylin and eosin and/or immunocytochemical staining and by scanning electron microscopy. Apoptosis and leakage of lactate dehydrogenase (LDH) were also assessed. All cells readily adhered to and spread on the surface of nitrocellulose membranes as well as coverslips, and the cells maintained the expression of digestive system-specific genes. No significant change was detected in apoptosis or leakage of LDH from cells grown on nitrocellulose membranes. These results suggested that nitrocellulose membranes have a suitable cytocompatibility towards human cells and that they might be used for tissue-engineering scaffolds. Moreover, we demonstrate an additional and underused property of nitrocellulose of specific relevance to microscopic imaging, as it can be rendered virtually transparent, thus the cells growing on such membranes can be observed directly under an optical microscope after staining.     

Arginine-specific cysteine proteinase from porphyromonas gingivalis as a convenient tool in protein chemistry.

RgpB, a cysteine proteinase produced by Porphyromonas gingivalis, exhibits proteolytic activity selectively directed against peptide bonds containing an arginine residue in the P1 position. Here we show that this enzyme can be used for very efficient and specific protein cleavage. RgpB is highly active even at high concentrations of denaturing agents, including urea (up to 6 M) and SDS (0.1%), both of them being commonly used for solubilization of insoluble proteins and peptides. Moreover, RgpB is able to digest polypeptide chains in buffers supplemented with 1% Triton X-100, 1% octyl or decylpyranoside, detergents employed for the enzymatic digestion of proteins transferred onto nitrocellulose membranes. These features render RgpB a suitable tool for use in protein chemistry.     

Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes

A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.     

Immunodetection with streptavidin-acid phosphatase complex on Western blots

A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated anti-antibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials.     

Multimembrane dot-blotting: a cost-effective tool for proteome analysis

The molecular profiles of protein expression from hundreds of cell lysates can be determined in a high-throughput manner by using fluorescent bead technologies, enzyme-linked immunosorbent assays (ELISAs), and protein microarrays. Although powerful, these tools are costly and technically challenging and thus have limited accessibility for many research groups. We propose a modification of traditional dot blotting that increases throughput of this approach and provides a simple and cost-effective technique for profiling multiple samples. In contrast to traditional blotting that uses a single membrane, we introduce blotting onto a stack of novel, thin, sieve-like membranes. These membranes have a high affinity for binding proteins, but have a lower capacity of protein binding compared to traditional (nitrocellulose) membranes. We compare the linear binding capacity and variability of these novel membranes with nitrocellulose membranes. Also, we describe the use of these membranes in a multilayer dot blot format for profiling mitogen-mediated signal transduction pathways in T cells.     

Immunological identification of aa3-type cytochrome oxidase in membrane preparations of the cyanobacterium Anacystis nidulans

Membranes were isolated from the cyanobacterium Anacystis nidulans by French press extrusion of lysozyme-treated cells. The membranes were solubilized with sodium dodecylsulfate and subjected to denaturing polyacrylamide gel electrophoresis. Separated polypeptides were transferred to nitrocellulose by Western blotting, and incubated with antibodies against aa3-type cytochrome oxidase of Paracoccus denitrificans; antibodies against subunits I and II, and against the holoenzyme, were used and gave pronounced complementary cross reaction with two of the Anacystis membrane polypeptides corresponding to molecular weights of approximately 55,000 and 32,000, respectively. From this we conclude that an aa3-type cytochrome oxidase is present in Anacystis nidulans as was previously suggested from spectral evidence (G.A.Peschek, Biochim.Biophys.Acta 635 (1981) 470-475), and that this enzyme is composed of at least two subunits with apparent homology to subunits I and II of the corresponding Paracoccus cytochrome oxidase.     

Comparison of different membranes for use in the colony-immunoblot technique

We compared five different supports (Whatman paper filters Nos. 1, 5, and 40, nitrocellulose, and Nylon 66) for their suitability in the colony-immunoblot (CIB) technique. Results indicate that Whatman No. 5 filter paper recovered 94-98% of the bacterial colonies tested, were more resistant to tearing than the other Whatman papers tested, and showed reduced cross-reactions as compared with nitrocellulose membranes. Whatman No. 5 filters are 20 times less expensive than the nitrocellulose membranes usually used in the CIB technique. We thus adopted the former for our ecological studies of the murine oral cavity.     

Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.     

A comparison of UV cross-linking and vacuum baking for nucleic acid immobilization and retention

The effectiveness of UV cross-linking and in vacuo baking for the immobilization and retention of DNA to various solid supports was investigated. Optimal immobilization treatments for supported and unsupported nitrocellulose and nylon membranes were: UV cross-linking at 254 nm with an exposure of 120 milliJoules/cm2, or baking in vacuo for two hours at 80 degrees C. UV-immobilized nitrocellulose-based membranes showed no increase in sensitivity when compared to baked membranes. An increase in sensitivity was observed for UV-immobilized nylon membranes as compared with baked nylon membranes in some instances, although this varied within lots of the membranes tested. Repeated strippings and heterologous reprobings resulted in loss of target DNA from UV-immobilized nylon membranes as compared to baked nylon membranes. Loss of target DNA from UV-immobilized nitrocellulose-based membranes due to repeated strippings and reprobings was even more pronounced. In vacuo baking of supported and unsupported nitrocellulose and nylon membranes was more effective for immobilization, and more importantly, for retention of target DNA through many reprobings of the same blot.     

Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A-gold

The method of electrophoretically transferring proteins from fixed and stained polyacrylamide gels onto nitrocellulose paper has been reevaluated. It is shown that the tedious destaining of gels is not necessary because Coomassie brilliant blue, although it binds tenaciously to nitrocellulose paper, does not reduce the transfer efficiency of proteins. However, its presence impairs the visibility of proteins as detected, for instance, by the immunogold technique. Therefore, a rapid method for the complete removal of the stain from the nitrocellulose paper after completion of the immunogold procedure was developed. Furthermore, it is shown that proteins from dried polyacrylamide gels can still be transferred onto nitrocellulose sheets with an efficiency of approximately 50% compared to proteins transferred from fixed gels.     

Southern transfer of native DNA using nylon membranes

Transfer of native or denatured DNA from gels or filter manifolds was compared using nylon or nitrocellulose membranes. The results were comparable when denatured DNA was used, but only nylon membranes were able to retain native DNA. Although retention of the native DNA was less efficient the bound DNA could be rapidly denatured in situ, avoiding the need to soak gels in alkaline denaturation solution and neutralizing buffer.     

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.