Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.     

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1 h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1 h with a detection limit of 0.2 ng/band.     

A method for rapid and sensitive negative staining of proteins in SDS‐PAGE using 2′,7′‐dichlorofluorescein

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.     

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.     

Quantification of proteins in the subnanogram and nanogram range: comparison of the AuroDye, FerriDye, and India ink staining methods

The usefulness of three sensitive dyes, AuroDye, FerriDye, and India ink, for the quantification of proteins and peptides bound to nitrocellulose paper has been assessed. In general, the staining intensity varies linearly with the logarithm of protein concentrations. The detection limit of small peptides (Mr less than 5000) is higher than that of large peptides and proteins, but the sensitivity is independent of the molecular weight. Oligopeptides of four or less amino acids either stain with very high detection limits or do not stain at all. The detection limit of proteins stained by AuroDye is approximately 1 ng, and in a number of cases even lower. The useful range for quantification of proteins extends to around 100 ng. The FerriDye and India ink staining methods are less sensitive and can be used to quantify proteins over a wide nanogram range. Among the methods tested, the India ink staining method has the highest protein to protein variation in sensitivity.     

Comparative analysis of an improved thioflavin-s stain, Gallyas silver stain, and immunohistochemistry for neurofibrillary tangle demonstration on the same sections.

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

A refined method for silver staining of nucleic acids fixed on nitrocellulose

A refined silver staining method was developed to stain nucleic acids fixed onto nitrocellulose membranes and nylon-based membranes. Approximately 4 ng RNA or DNA can be stained with this method with no protein interference. This method involves simple repetition of immersions of membranes in three solutions prepared from common chemicals. The total staining time is less than 30 min.     

A rapid single step staining technique for DNA analysis by flow microfluorimetry

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.     

Interferometry in flow to sort unstained X- and Y-chromosome-bearing bull spermatozoa

BACKGROUND: It was found earlier that the difference in volume between unstained X- and Y-chromosome-bearing sperm heads could be detected using interference microscopy in visible light. This could be the basis for an alternative to the conventional method to sort X and Y sperm, which uses DNA staining and ultraviolet (UV)-excitation that may be harmful to sperm cells. A novel technique is introduced combining interferometry with flow cytometry. MATERIALS AND METHODS: Interference optics were built into an existing flow cytometer/cell sorter and used to sort fresh unstained bull sperm cells on the basis of their head volume. Sorted fractions were stained with a DNA stain, and reanalyzed using the conventional method. RESULTS: Purities between 60-66% were found in both X- and Y-enriched fractions. It was possible to sort up to 300 cells per second. The system was found to be less sensitive to the orientation of sperm cells than the conventional method. CONCLUSIONS: Interferometry can be combined with flow cytometry and used to obtain significantly enriched fractions of X- and Y-bearing sperm without staining and UV light. Sorting speeds and purities at this point, however, are much lower than with the conventional method.     

微量蛋白检测中不同银染方法的比较与分析

随着生物化学技术的不断发展,作为检测SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中微量蛋白的银染方法也在不断改进和发展.采用4种不同的银染方法检测不同含量的牛血清白蛋白,结果显示单纯的银染过程中如果使用戊二醛固定会使蛋白检出更快速灵敏,而结合考马斯亮蓝的复合银染则较单纯银染灵敏度提高了5～7个数量级.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

Modification of the method for staining DNA with basic dyes]

The author studied the mechanisms and the applicability in histochemistry of the sodium bisulfate treatment with subsequent toluidine and methylene blue staining after Felgen's hydrolysis. Bisulfite treatment proved to increase the reaction intensity 11/2-fold; the stain is bound stoichiometrically. Toludidine blue results in a metachromatic and anisotropic staining of the cell nuclei. The method is recommended as a sensitive test for DNA in cytochemical investigations and for the study of dichroism of the DNA-containing structures.     

A New, Rapid, Microwave-Stimulated Method of Staining Melanocytic Lesions

A new and sensitive method of staining melanocyte lesions is described. Tissue sections covered by a solution of colloidal silver nitrate are exposed to microwaves for 45 sec in a domestic oven to produce clean, crisp staining of melanocytes and melanoma cells, often showing long delicate dendritic cell processes. The staining technique does not stain other pigments or argyrophilic tissues and is shown to be more sensitive than the standard Masson-Fontana procedure.     

A new, rapid, microwave-stimulated method of staining melanocytic lesions

A new and sensitive method of staining melanocytic lesions is described. Tissue sections covered by a solution of colloidal silver nitrate are exposed to microwaves for 45 sec in a domestic oven to produce clean, crisp staining of melanocytes and melanoma cells, often showing long delicate dendritic cell processes. The staining technique does not stain other pigments or argyrophilic tissues and is shown to be more sensitive than the standard Masson-Fontana procedure.     

An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears.

We developed a novel method for the detection of Mycoplasma hominis from vaginal swabs using an indirect immunofluorescence technique. It is a rapid and simple method that can be finished in only 5 hr and is more sensitive than the usual culture isolation method. The indirect immunofluorescence method was applied to vaginal smears from 193 healthy women and 33.7% gave a positive test. This value was much higher than that (11.4%) obtained from the same specimens by the culture method. When vaginal smears were subjected to Papanicolaou staining after the indirect immunofluorescence method, the specific immunofluorescence of the epithelial cells was located exactly at the sites of granular aggregates stained with Papanicolaou stain. A histological examination by Papanicolaou staining showed that the incidence of inflammation seems to be slightly higher in M. hominis-carriers than in non-carriers.     

Immunocytochemical detection of sulphonylated DNA in tissue sections

Summary We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Immunocytochemical detection of sulphonylated DNA in tissue sections. An alternative to Feulgen staining of DNA

We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2'-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Comparison of the sensitivity of the indirect,antibody-conjugated and the triple-bridge immunoperoxidase methods for immunohistochemical detection of carcinoembryonic antigen

Summary A comparison between two immunoperoxidase staining procedures, a triple-bridge and an indirect, antibody-conjugated method, was made to determine the relative sensitivity of each technique in the detection of carcinoembryonic antigen (CEA) in routine tissue sections. The enzyme-antibody conjugates for the indirect procedure were prepared according to the method of Nakane and Kawaoi (1974). The indirect, antibody-conjugated method, proved to be slightly more sensitive than the triple-bridge by two criteria. First, CEA could be localized using higher dilutions of the primary antiserum by the indirect technique, and secondly, tissues were shown to stain for CEA in specimens with lower tissue CEA levels by the indirect procedure than by the triple-bridge method. The preparation of enzyme-antibody conjugates is a relatively simple procedure and, in addition, the conjugates will remain stable when kept frozen or at 4°C. Background staining due to non-specific interaction of the conjugate with the tissue can be eliminated easily by incubation with normal serum. These results indicate that the indirect, antibody-conjugated method can be used to enhance the staining of CEA in routine tissue sections.Supported in part by NIH contract NCI-NO1-CB-84257     

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.