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Although proteins can be transferred and bound to a membrane with several different methods such as capillary transfer, electroblotting, etc., only the "shaker/incubation" method is commonly used for visualization of the proteins. We have tested an apparatus for the immunofiltration of solutions through nitrocellulose membrane which greatly accelerates the kinetics for the visualization of proteins. As a model system, avidin, bound on nitrocellulose membrane, was detected with 5-min filtering steps of a 1:2000 dilution of ascites of murine monoclonal antibody against avidin followed by a 1:5000 dilution of goat anti-mouse IgG-horseradish peroxidase and 0.5 mg/ml chloronapthol in the presence of 0.01% peroxide. The same solutions used with 5-min incubation steps with the shaker/incubation method could not detect avidin at almost 10 times that amount. Further studies with monoclonal antibodies specific for native C-reactive protein and modified-CRP, 15.1D6 and 13.3H12 respectively, showed that immunofiltration did not result in altered specificity compared to the shaker/incubation method. Also, data are presented showing the advantages of a 10-slot top for the immunofiltration of solutions through distinct areas of a membrane.
A method is described for the use of nitrocellulose powder as a solid phase in a chromatographic procedure, for the immunoaffinity isolation of proteins. Two different immunoglobulins (Igs), anti-Datura innoxia lectin and anti-tyrosinase, were coupled to particulate nitrocellulose. A single step was then needed for purification to homogeneity of both D. innoxia lectin and mouse tyrosinase. Chaotropic and acidic agents proved to be effective in eluting antigens from Ig-nitrocellulose columns. The binding capacity of particulate nitrocellulose was around 3 mg Ig per milliliter of nitrocellulose, while the purification yields of the two proteins investigated under various eluting conditions were higher than 75%. The applicability of this method in the identification of metabolically labeled proteins in crude extracts is also demonstrated. Purification of proteins by affinity chromatography on their specific Igs linked to nitrocellulose matrices could be performed in both batch and column. The major advantages of this new method for purification of proteins are its rapidity, the reusability of the affinity matrices, and the high yields of purified protein obtained. The method could be seen as an alternative to the widely used immunoprecipitation technique.
Dopachrome isomerase, which mediates the conversion of dopachrome to dihydroxyindoles, is an enzyme affiliated with the melanogenic pathway found in most vertebrates and invertebrates. We have recently reported an activity-staining procedure for detecting this enzyme after electrophoresis on tyrosinease-embedded polyacrylamide gels using dopa [K. Nellaiappan, G. Nicklas, and M. Sugumaran, (1994) Anal. Biochem. 220, 122-128]. The usefulness of this method is limited to the availability of good quality of tyrosinase. To overcome the limitations of nonavailability as well as batch to batch variation in the specific activity of commercially available tyrosinase which will affect the staining, we have developed a new stain technique using chemically made dopachrome for the detection of dopachrome isomerase. The new method employs the use of dopachrome generated in situ by the oxidation of dopa with periodate. Dopachrome isomerase initially appears as a bluish-purple (melanochrome) band against an orange-red background. Eventual transformation of melanochrome to dark insoluble melanin polymer produces a black band corresponding to dopachrome isomerase activity. Continuous washing of the gel clears the background. Usefulness of this technique to stain dopachrome isomerase (a) in different organisms and (b) on polyacrylamide gels, agarose gels, and nitrocellulose membranes is demonstrated. In addition, conditions to stain the enzyme on nitrocellulose membranes using mushroom tyrosinase are described. Comparison of the effectiveness of tyrosinase versus periodate staining procedure reveals that the former method is superior to the latter in detecting dopachrome isomerase. The periodate method overcomes the problems associated with the quality of commercially available tyrosinase and its nonavailability in certain regions.
Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose. This compound has a biotin as one functional group and a nitrilotriacetic acid as the other. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.
A rapid and simple electrophoretic separation method on nitrocellulose membranes by a single run of glycosaminoglycans, mainly slow-moving and fast-moving heparins, dermatan sulfate, and chondroitin sulfate, is reported. Different dyes, azure A, toluidine blue, and Alcian blue, were used to stain the nitrocellulose strips, and azure A showed the highest sensitivity. The mobility for slow-moving heparin was 0.00 cm/A/min, for fast-moving species about 2.87 x 10(-3) cm/A/min, for dermatan sulfate 4.00 x 10(-3) cm/A/min, and for chondroitin sulfate about 5.75 x 10(-3) cm/A/min. Calibration curves were determined by the increasing amounts (from 0. 1 to 2.5 microg) of single glycosaminoglycan species. The curves demonstrated a linear relationship between the amount of glycosaminoglycans (slow-moving and fast-moving heparin, dermatan sulfate, and chondroitin sulfate) and the optical density at 600 nm calculated by densitometric scanning. The limit of detection for fast-moving heparin, dermatan sulfate and chondroitin sulfate was about 0.2-0.3 microg, while that for slow-moving heparin was 0.1 microg. Hyaluronic acid, keratan sulfate, and highly sulfated chondroitin sulfate from shark cartilage were also separated electrophoretically on nitrocellulose. No difference in mobility was detected for chondroitin sulfates with different structure and physicochemical properties. Hyaluronic acid has about the same mobility as that of dermatan sulfate, while keratan sulfate has a mobility intermediate between those of dermatan sulfate and chondroitin sulfate (about 4.75 x 10(-3) cm/A/min). Glycosaminoglycans extracted and purified from bovine aorta and bovine lung were submitted to electrophoresis on nitrocellulose and the amount of each polysaccharide species was calculated by densitometric scanning. By this analysis bovine aorta glycosaminoglycans consist of about 11% fast-moving heparin (heparan sulfate), 23% dermatan sulfate, and 65% chondroitin sulfate. Bovine lung polysaccharides are composed of about 16% slow-moving and 46% fast-moving species (62% heparin), 15% dermatan sulfate, and 22% chondroitin sulfate.
We have analyzed the efficacies of seven different organic solvents, 13 organic bases, and 17 detergents to dissociate electroblotted proteins from nitrocellulose. Most efficient were a 1% piperidine-40% acetonitrile mixture and 1% concentrations of either cetyltrimethylammonium bromide or several polyoxyethylene and zwitterionic detergents at pH > or = 8.5. In general, detergent-promoted elution varied with pH (8.5-->12.0) and temperature (37-->65 degrees C) in a detergent-dependent and protein-dependent manner, making for unpredictable results of any combination. However, Zwittergent 3-16, as the major exception, eluted proteins of different sizes and properties almost equally well at pH 8.5. This preferred effect is not enhanced by addition of organic solvent. In fact, detergent/acetonitrile mixtures were generally less efficient for protein elution. Zwittergent 3-16 (1% in 100 mM NH4HCO3) proved to be the most powerful additive for one-step enzymatic digestion of nitrocellulose-bound proteins, both in terms of highest recoveries (including endoproteinase Lys-C digestions) and general applicability, a major improvement over the use of Tween 80 and hydrogenated Triton X-100. The new digest procedure takes just 2 h to complete and requires 5 ng protease per 1 microliter buffer per 1 mm2 of nitrocellulose; relatively large pieces of membrane (> or = 100 mm2) can be successfully processed. The same detergent is a satisfactory additive for digestion of polyvinylidene difluoridebound proteins as well. Moreover, Zwittergent 3-16 is fully compatible with the usual downstream procedures. It is uv (OD214) transparent and does not interfere with high-performance liquid chromatography in any other way. Apparent incompatibility with direct (i.e., "prechromatography") matrix-assisted laser-desorption ionization time-of-flight mass spectrometry can be either reduced by methanol washing of the dried sample and activation of a low-mass gate or eliminated by a simple microliter cleanup procedure.
Proteins absorbed directly onto nitrocellulose membranes were stained with amido black, ponceau S, colloidal silver, or Coomassie blue and solubilized in dimethyl sulfoxide and the absorbance was measured spectrophotometrically. The optimal wavelength of each dye/protein/nitrocellulose solution was found to be at 625, 529, 420, and 600 nm, respectively. A linear relationship was found between the protein concentration and absorbance at the appropriate wavelength for all the stains with individual purified proteins or protein mixtures. Protein (0.2-0.8 microgram) can be determined with the colloidal silver and 2-30 micrograms with the other stains. Coomassie blue produced variable background staining of the nitrocellulose and is therefore not recommended. Proteins transferred electrophoretically to nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel were also stained with the above dyes and solubilized in dimethyl sulfoxide. Amido black was the most sensitive stain, detecting proteins in the range of 1-10 micrograms. Components of the gel interfered with silver staining.
Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. Detecting the absorbance of colorimetric stains is limited by the molar extinction coefficient of the product, however. Development of light-emitting complexes should improve detection sensitivity, but fluorescent labels described to date modify free amino, carboxyl, or sulfhydryl groups often rendering proteins unsuitable for further analysis. Bathophenanthroline disulfonate (BPSA) forms a luminescent europium (Eu) complex that reversibly binds to proteins and nucleic acids. Analysis of charge-fractionated carrier ampholytes and synthetic polymers of different L-amino acids indicates that protein binding is chiefly through protonated alpha- and epsilon-amino side chains. Proteins or nucleic acids immobilized to a nitrocellulose or polyvinyl difluoride membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubated with the lanthanide complex at acidic pH. Membranes are rinsed, illuminated with UV light and the phosphorescence of BPSA-Eu is measured at 590 to 615 nm using a CCD camera or spectrofluorimeter. The linear dynamic range of the stain is 476- and 48-fold for protein and DNA, respectively. A strong chelating agent such as ethylenediaminetetraacetic acid combined with a shift to basic pH (PH 8-10) elutes BPSA-Eu from the membrane. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, lectin staining, and mass spectrometry.
High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.
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A semidry electrophoretic transfer method was developed for efficient electroblotting of proteins separated by acid-urea polyacrylamide gel electrophoresis (AU-PAGE). Model polypeptides ranging from 1.8 to 21.5 kDa were used to test transfer parameters that included time of transfer, power settings, transfer solutions, and membrane type. Optimized conditions were identified which allowed for transfer efficiencies of 70-100% following 5-15 min of applied current. The best transfer solution was 5% acetic acid, the same solvent used for electrophoresis. Therefore, acid-urea gels could be subjected to electrophoretic transfer without a soaking step, thereby reducing loss of band resolution and eliminating leaching of protein from the gel. The method was shown to be applicable to Western blot analysis of rat neutrophil defensins.
An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simultaneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.
Linker histones from grapevine were purified by electrophoretic methods (SDS-PAGE and electrotransfer onto a nitrocellulose sheet). Individual linker histones were recovered after nitrocellulose solubilization by acetone. The proteins precipitated after this treatment were used as antigen for subsequent immunizations of mice. Such purified immunogens were injected into mice with dimethyl sulfoxide, as the presence of nitrocellulose-protein complexes in the antigen pellets after the acetone treatment was suspected. The resulting antisera were specific to the injected antigens after only the first immunization and could be used as specific probes in immunohistological studies. Our approach seems more efficient (in using less antigen and obtaining a faster response) than the classical procedure recommended for histones in general (B. D. Stollar and M. Ward, 1970, J. Biol. Chem. 245, 1261-1266) and other methods that use free linker histone as immunogen.
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We describe a method for estimating ligand binding to a macromolecular sample under conditions where this binding is of low affinity and must be measured under equilibrium conditions, without removal of the unbound ligand. The method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the difference between the (total) ligand in the concentrated sample and the (free) ligand in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to separate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of bound ligand becomes significant, even under low-affinity binding conditions). We applied this technique, using Centricon 10 (Amicon) devices, to several cases (soluble proteins, intact membranes, detergent-solubilized proteins, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose filters (nonspecific ligand adsorption and protein denaturation).