A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate

A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 microgram/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

A high-affinity reversible protein stain for Western blots

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.     

Silver Enhancement of Nickel-Diaminobenzidine as Applied to Single and Double Immunoperoxidase Staining

A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation.     

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system.

Proteins are biotinylated after immobilization on nitrocellulose sheets by reaction with a biotinyl-succinimide ester. The biotinyl residues are visualized by streptavidin-peroxidase-based detection systems either by deposition of a colored formazan dye or by enhanced chemiluminescence (ECL), the latter being 10-fold more sensitive. The sensitivity of the staining procedure is dramatically improved by the inclusion of the reporter deposit technique into the staining procedure: the initially bound peroxidase generates phenolic radicals from biotinyltyramide, enhancing the number of biotinyl residues in the vicinity of the first biotinylation site. Thus the detection limit is lowered to 1 pg of protein with the ECL detection. The new method is compared with silver stain and immunochemical staining in Western blots and furthermore its suitability is demonstrated for 2-D gel electrophoresis.     

Wafer embedding: Specimen selection in electron microscopic cytochemistry with osmiophilic polymers

Synopsis A new wafer embedding procedure is described that permits light microscopic screening of embedded tissue prior to ultrathin sectioning. It is particularly valuable when used on specimens obtained with an automatic sectioner and treated cytochemically to obtain visible intermediate or visible and electron opaque final reaction products. Aldehyde-fixed tissues are cut into sections with an automatic sectioner, incubated cytochemically including osmication if required, then embedded in epoxy resin between fluorocarbon-coated coverglasses which are supported by a platform specially designed for this purpose. The resultant wafer, less than 0.2 mm thick, is examined by light microscopy for optimal areas of cytochemical reaction and desirable structural features. Such areas are cut out and glued to blank blocks with fast curing cyanoacrylate cement for subsequent ultrathin sectioning. The usefulness of this technique is demonstrated by the location of: (1) esterase-positive lysosomes in kidney and trigeminal ganglia; (2) palatal sensory endings stained for acetylcholinesterase; and (3) phagosomes arising from the resorption of horseradish peroxidase tracer by the cuboidal parietal epithelial cells of Bowman's capsule in the male mouse.     

Axonal Polypeptides Cross-Reactive with Antibodies to Neurofilament Proteins

Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross-reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000, and 70,000 NFPs (200K, 140K, and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase-conjugated indirect antibody procedure, however, differences were detected in the cross-reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and, thus, was suitable for the detection of cross-reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti-200K, anti-140K, or anti-70K was applied to overloaded two-dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross-reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Titers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.     

Fluorescamine, a fluorescence probe for amino groups in histochemical studies of plant cells and the effect of mercury fixation

Synopsis When fixed in mercuric chloride solutions and stained with Fluorescamine, histological plant specimens emit a strong fluorescence. The fluorophore distribution is topologically identical to the staining pattern revealed by visible light methods for nucleoproteins, but the fluorescence mode of viewing preparations gave greater sensitivity and contrast than transmitted light absorption methods. The parameters that influence the formation of the fluorescent image in plant cells are discussed. The results obtained indicate that the mercury-Fluorescamine reaction is an ideal histochemical procedure for collecting qualitative and analytical information on plant nuclei and on the changes of nucleolar architecture that occur during the cellular developmental cycle.     

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.     

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.     

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.     

Immunoaffinity layering enhances the sensitivity of antigen detection on nitrocellulose strips

A simple strategy to remarkably increase the sensitivity of detection of antigens applied as dot or western blot on nitrocellulose membrane using human serum albumin as model antigen has been described. This involves subjecting the antigen bearing nitrocellulose strips to multiple incubation cycles with primary antibody and enzyme conjugated secondary antibody prior to staining for enzyme activity. The sensitivity of detection could be increased up to a thousand fold after three incubation cycles. Aggregation of human serum albumin could be detected by the multiple incubation procedure at very low protein concentration after electrophoresis and transfer onto nitrocellulose.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

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.     

Sensitive assay of cytochrome P450scc activity by high-performance liquid chromatography

We have developed a simple procedure for analyzing the reaction intermediates and product of the cholesterol side-chain cleavage system by high-performance liquid chromatography with uv absorption monitoring. After the cholesterol side-chain cleavage system had been incubated and the reaction then halted by heat treatment, the product was converted into 3-one-4-en steroid showing intense absorption at 240 nm upon reaction with cholesterol oxidase. The converted steroids were then analyzed by normal-phase HPLC. In consequence, the catalytic activity of the reconstituted adrenocortical cytochrome P450scc system was readily assayed with a sensitivity more than 10-fold higher by this conversion. Also, it was shown that 22R-hydroxy-cholest-4-en-3-one could serve as a good substrate for cytochrome P450scc and that the 20R,22R-dihydroxy derivative could be clearly detected as a reaction intermediate in the reconstituted system.     

Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immobilized on nitrocellulose

The nitrocellulose binding assay was used for quantitative studies on the cytochemical reactions for the three enzymes most frequently used in immunocytochemistry. The results show a linear relationship between the amount of enzyme immobilized on nitrocellulose and the amount of the enzyme reaction product. The similar course of the formation of the reaction product after DAB/H2O2 staining for peroxidase immobilized on nitrocellulose and for immunoperoxidase labeled cells indicates a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. Furthermore, a mild acid treatment for the abolition of endogeneous peroxidase activity in tissues and cells applicable to immunoperoxidase staining procedures is proposed.     

Polyacrylic polyhydrazides as reagents for detection of glycoproteins

Glycoproteins immobilized on membranes can be detected with high selectivity and sensitivity by the four-step procedure described in this work. The glycoproteins are first oxidized by sodium periodate and then polyacrylic polyhydrazides are coupled to the aldehyde groups generated in the sugar part of the glycoproteins. In the third step, a glycoenzyme, such as horseradish peroxidase, is coupled to the remaining hydrazide groups on the polymer through the aldehydes formed in its glycan chains. In the last step, the visualization of glycoproteins is achieved through the reaction product of the bound glycoenzyme. The sensitivity of the glycoprotein detection is most critically dependent on the hydrazide reagent. Thus, dihydrazides were not satisfactory, a trihydrazide was better, and polyhydrazides were the best. Two different polyhydrazides were used. One was based on acrylamide and the other on N-acryloyl-tris(hydroxymethyl)aminomethane. The second one proved to be superior because it gave higher sensitivity with no detectable background staining. We have also investigated the influence of various reaction conditions on staining of glycoproteins having oligomannose and N-acetyllactosamine type glycan chains. Some of them, invertase and fetuin, could be detected with sensitivity similar to that of silver staining in gels and colloidal gold staining on the membranes. The detection of small quantities of Endo H-deglycosylated glycoproteins was possible under standard conditions only if several N-acetylglucosamine residues remained bound to the protein.     

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.     

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.