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.     

Double staining to increase the sensitivity of protein detection in polyacrylamide gels.

As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis gels with Pro-Blue (colloidal blue stain) followed by silver staining--yields an extremely sensitive, nonspecific protein stain. On average, this double-staining technique resulted in a 40-fold increase in sensitivity and intensity vs. silver stain alone. This is a tremendous return for a small investment in additional time and materials.     

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.     

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.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

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.     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

Staining of Paneth Cells with Best's Carmine After Methylation

When sections are methylated (cone. HC1, 0.8 ml in absolute methanol, 100 ml; at 58 C) prior to staining with Best's carmine, the granules of Paneth cells of man, rat and mouse stain a bright red, but they do not stain at all with this stain without prior methylation. With paraffin sections after neutral formalin fixation, the required 2-hr methylation did not prevent the staining of neutral mucosubstances and glycogen, but after methylation for 12 hr, these substances no longer stained although the reaction of the granules of Paneth cells became still more intense. The advantages of this staining technique are: (1) There is good contrast because the background stains faintly and, of the structures in the intestinal wall, only eosinophilic leukocytes and a part of the collagen fibrils stain in addition to the granules of Paneth cells. (2) The result is more reliable and the staining easier to perform than with the majority of other techniques, since no differentation is necessary. The method is especially suited for detecting Paneth cells in pathological conditions and in altered tissues or areas in which these cells are scanty.     

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.     

Elastin V: Syntheses, Characterization, and Staining Properties of Amidol Black I

One of the monoazo dyes reported by us in an earlier study (Lillie et at. 1972), amidol brown NAP, yielded an oxidation product, amidol black I, which was an excellent elastin stain. The present study revises the synthesis of amidol black I to a more convenient form giving much larger yields. Briefly, diazotized 4-nitro-2-aminophenol is azo-coupled into 2, 4-diaminophenol (amidol) and the product is oxidized to a quinoneimine with nitric. The product stains elastin black from an acid alcohol bath according to a Taenzer-Unna (1890) orcein type technic. The stain may be combined with a Van Gibson collagen stain or with an oil red O fat stain. The synthesis is presented in sufficient detail to permit its repetition in a hospital pathology laboratory.     

Analysis of the Microbiota of Black Stain in the Primary Dentition

Black tooth stain is a characteristic extrinsic discoloration commonly seen on the cervical enamel following the contour of the gingiva. To investigate the relationship between black tooth stain and the oral microbiota, we used 16S rRNA gene sequencing to compare the microbial composition of dental plaque and saliva among caries-free children with and without black stain. Dental plaque and saliva, as well as black stain, were sampled from 10 children with and 15 children without black stain. Data were analyzed using the pipeline tool MOTHUR. Student’s t-test was used to compare alpha diversities and the Mann-Whitney U test to compare the relative abundances of the microbial taxa. A total of 10 phyla, 19 classes, 32 orders, 61 families and 102 genera were detected in these samples. Shannon and Simpson diversity were found to be significantly lower in saliva samples of children with black stain. Microbial diversity was reduced in the black stain compared to the plaque samples. Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Tannerella and Treponema were more abundant and Campylobacter less abundant in plaque samples of children with black stain. Principal component analysis demonstrated clustering among the dental plaque samples from the control group, while the plaque samples from the black stain group were not and appeared to cluster into two subgroups. Alterations in oral microbiota may be associated with the formation of black stain.     

Inhibin may be involved in negative feedback in the prairie dog (Cynomys ludovicianus)

The changes in inhibin immunostaining in the gonads during the annual reproductive cycle of both sexes of the prairie dog are described. No inhibin immunostaining was found in primary or secondary follicles of the ovary. Theca and granulosa cells of preovulatory Graafian follicles found in January and February stained for inhibin. Corpora lutea of both pregnant and non-pregnant females stain more densely for inhibin than follicles. Inhibin staining is present in luteal cells for at least 4 months during regression, longer than detectable progesterone is secreted. Sertoli cells in the testes do not have inhibin immunostaining during recrudescence. These cells show light immunostain for inhibin during peak spermatogenic activity in January and February but stain more deeply during early regression of the testis. Stain is gradually lost in the next 4-5 months as the tubules close. Leydig cells and germ cells do not stain for inhibin at any stage of the annual cycle but interstitial cells and tunic cells stain during the breeding phase. The presence of immunochemical staining for inhibin in prairie dog gonads during regression suggests that inhibin is part of a negative feedback complex that includes progesterone in the female and testosterone or another androgen in the male. Negative feedback during regression may also cause gonadal inactivity.     

The Gram Stain after More than a Century

The Gram stain, the most important stain in microbiology, was described more than a century ago. Only within the past decade, however, has an understanding of its mechanism emerged. It now seems clear that the cell wall of Gram-positive microorganisms is responsible for retention of a crystal violet:iodine complex. In Gram-negative cells, the staining procedures damage the cell surface resulting in loss of dye complexes. Gram-positive microorganisms require a relatively thick cell wall, irrespective of composition, to retain the dye. Therefore, Gramstainability is a function of the cell wall and is not related to chemistry of cell constituents. This review provides a chronology of the Gram stain and discusses its recently discovered mechanism.     

Histochemical Identification of Basic Proteins with Biebrich Scarlet at Alkaline pH

Staining at graded alkaline pH levels with the sulfonated dye, Biebrich scarlet, shows basic proteins in various histologic sites, and differentiates the sites according to their relative basicity. Certain structures stain at pH 6.0 but not at 8.0 or above. Others stain maximally up to pH 93 and a few stain strongly as high as pH 103. The most strongly basic sites resist more than the others the destruction of acidophilia by nitrosation, acetylation or exposure to formaldehyde.     

The demonstration of B cells in pancreatic islets with Orcein

Summary Orcein stains granules in pancreatic islet cells selectively. The localization of Orcein-positive cells within islets differs from that of Grimelius-stained cells, but corresponds to the B cell type differentiated by Aldehyde Fuchsin.Usually there appear to be fewer Orcein-positive cells than Aldehyde Fuchsin-positive ones. This indicates either that Aldehyde Fuchsin is a more sensitive stain for B cells or that Orcein is a more selective stain for a B cell type subpopulation. The rationale of the Orcein reaction in B cells seems to depend on the oxidation of disulphide bonds present in insulin and its precursors rich in cystine.     

Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study

Gerodontology 2012; doi: 10.1111/j.1741‐2358.2011.00522.x
Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study Objectives: To determine the ability of eight denture cleansers to remove and inhibit tea‐stain build‐up on acrylic resin. Materials and methods: In the stain removal study, Perspex^® (cast heat polymerised resin) specimens previously soaked in saliva were stained using multiple exposures of chlorhexidine and tea solutions. Specimens were exposed for 1 min to one of the eight denture cleansers for five cycles, washed and dried and their optical density read on a uv/vis spectrophotometer at 295 nm. In the stain inhibition study, clear specimens were exposed to saliva followed by cleansers then tea solution, for five cycles. The build‐up of stain at each cycle was measured, and differences in optical densities from baseline were calculated. Results: All denture cleansers were significantly more effective than water in removing stain (p < 0.05). There were significant differences in cleaning ability between cleansers (p < 0.001), Dentural^® and Kleenite^® were particularly effective. The stain inhibition experiment showed that most cleansers were significantly more effective than water in inhibiting stain (p < 0.05). There were significant differences in inhibition ability between cleansers (p < 0.01). Kleenite^® and Equate were particularly effective. Conclusions: All denture cleansers had a capacity to remove stain and most had an inhibitory effect on staining. Kleenite^® was particularly effective in controlling stain formation.     

Feulgen, iron-propionocarmine and cupra-ammonium in preparing algal chromosomes for light microscopy.

A method for obtaining algal chromosomal preparations is described employing the Feulgen method for DNA staining, Fe-propionocarmine as an enhancing stain, and cupra-ammonium to remove cell wall material. Fe-propionocarmine applied as a gradient to the side provides cells stained with the Feulgen stain alone or with the Feulgen Fe-propionocarmine stain, thereby facilitating useful comparison. Where dilute the Fe-propionocarmine enhances nuclear staining without staining orthe organelles; where more concentration it also stains the nucleolus, spindle, spindle polar bodies, pyrenoid and protoplast. Treatment with cupra-ammonium, to remove polysaccharide wall material, followed by neutralization with propionocarmine, enables thinner squashes and better chromosome spreads without loss of differential staining. Preparations mounted in euparal are long-lasting.     

Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Identification and elimination of heterophilic antibody interference during antibody pair screening

A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30 min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40 ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole-zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory.