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.     

Immobilization of DNA on microporous membranes using UV-irradiation]

Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.     

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.     

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.     

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.     

Western blots using stained protein gels.

A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.     

An Improved Solid-Phase Bioassay for Evaluation of Neurotrophic Factors

Abstract: A bioassay system originally described by other investigators was improved to evaluate the biological activities of neurotrophic factors (NTFs) and is referred to as a solid-phase bioassay (SPB). The principle and an outline of the SPB are as follows: (a) Test samples containing NTFs are applied to polyacrylamide gel electrophoresis (PAGE) of the sodium dodecyl sulfate (SDS) or two-dimensional type and then transferred onto nitrocellulose membranes, (b) neurons are cultured on the protein-blotted membranes, and (c) the distributions of the surviving neurons are estimated following fixation and clarification of the nitrocellulose membranes. The rationale is that neuron survival will be restricted to the migration positions of the NTF(s). We used nerve growth factor (NGF) as a NTF and sympathetic neurons of 10- to 12-day-old chick embryos as NGF-responsive neurons. Neuronal survival was observed in the areas coinciding with the positions of NGF migration on the membranes in both systems following SDS-PAGE and two-dimensional PAGE. These results indicate that the SPB can identify the positions of NGF-like molecules on nitrocellulose membranes. Using this system, we found active entities, with molecular masses of ∼ 100–200 kDa, different from NGF in crude extracts of mouse submaxillary glands. The SPB developed is considered to be a useful tool for obtaining information on the physicochemical and/or biological properties of putative NTFs in crude samples.     

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.     

Negative purification method for the selection of specific antibodies from polyclonal antisera

We developed a protocol to remove non-specific antibodies from polyclonal antisera by adsorption on non-target antigens immobilized on nitrocellulose membranes. This "negative" purification method is simple and provides better immunoreagents than the blocking of nonspecific antibodies in solution or the enrichment of specific antibodies on nitrocellulose membranes. For routine applications, this method is quicker and cheaper than the purification protocols based on selective precipitations and affinity chromatography.     

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.     

Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet

The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.     

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.     

Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor

An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.     

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.     

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.     

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.     

On the electrotransfer of polypeptides from gels to nitrocellulose membranes

The conditions which affect the elution of polypeptides from polyacrylamide gels by electrophoresis and polypeptide-nitrocellulose interactions have been studied. The rate of elution of polypeptides from a 15% sodium dodecyl sulfate-polyacrylamide gel is dependent on the molecular weight of the individual polypeptides, which is in agreement with the results of W. N. Burnette (Anal. Biochem. 112, 195 (1981)). We also observed that current density affects the rate of elution. Polypeptides smaller than 20,000 daltons pass through pores of 0.45 microns, but not through the pores of 0.1-microns nitrocellulose membranes during electrophoresis. The nonionic detergent NP-40 inhibits the binding of polypeptides to nitrocellulose and removes prebound polypeptides from the membranes. Amido black and Coomassie blue staining and destaining processes do not remove the bound polypeptides from the membranes, but may affect the antigenicity of polypeptides. Polypeptides immobilized on nitrocellulose can be stored at -70 degrees C for future use.     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.