Staining protein in isoelectric focusing gels with fast green

A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated.     

Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis.

The properties of amido black 10B (C.I. 20470), Coomassie blue R (C.I. 42660), and fast green FCF (C.I. 42053) as protein stains, along with a few comments on Coomassie blue G (C.I. 42655), are presented and dye impurities and their effects on protein-dye binding within gels are discussed. All three dyes produced metachromatic effects with some proteins. Problems encountered with long-term stability and fixation of certain maize seed proteins are reported along with procedures for overcoming them. The low solubility of Coomassie blue R in trichloroacetic acid prevented maximum staining and destaining within a reasonable time, whereas other solvents allowed diffusion of some proteins during staining. Coomassie blue R binds to proteins in much higher amounts than do amido black and fast green, which accounts for its sensitivity in detection of protein bands in gels. Procedures for obtaining maximum contrast with photographs are also outlined.     

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.     

Staining of histones on polyacrylamide gels with amido black and fast green

The absorption characteristics and stability of amido black-histone complexes are shown to be highly dependent on the kind of histone involved. The potential of amido black for qualitative analyses of histone complements, and some risks in its use for quantitation are indicated. The absorption characteristics of fast green also depend to some extent on the kind of histone it is bound to. Fast green occurs in blue as well as green forms in histone complexes, and the proportion of blue to green forms may vary according to the kind of histone and the extent of destaining. In quantitative analyses based on fast green staining, measurements of the blue form should be preferred since complexes of different histones with the blue form are all relatively stable.     

Staining of phospho-proteins on acrylamide gel electropherograms

The paper describes a method for staining the phosphate moiety of phospho-proteins after electrophoresis on polyacrylamide gels. The method depends on hydrolysis of the phospho-protein phosphoester linkage using dilute base in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is finally stained with the basic dye, methyl green, dissolved in dilute acetic acid. After destaining, a green band is observed on the gel at the locus of the phospho-protein. One nanomole of protein-bound phosphate is detectable.     

Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.     

Staining Rickettsiae in Yolk-Sac Cultures

Rickettsiae in yolk sacs are not stained well by the Macchiavello technique, and experiments were undertaken to understand the mechanisms involved. It was found that the citric acid destaining step was not effective and that most of the basic fuchsin was lost from the rickettsiae during the application of methylene blue, another basic dye. A staining technique was then evolved with carbol basic fuchsin in pH 7.45 phosphate buffer (0.4% dye, 0.4% phenol, 0.07 M buffer), followed directly by 0.8% aqueous malachite green oxalate. This technique worked well for R. mooseri, R. prowazeki, R. rickettsii, R. akari, and R. buretii, but for R. tsutsugumushi a modification was needed, whereby 4% aqueous Fe(NO₃)₃·9H₂O was used as destaining solution, and 0.5% aqueous fast green as the counter-stain.     

Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate.

The utilization of Fe²⁺-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The first procedure is used in standard disc electrophoresis and involves fixing the protein with trichloroacetic acid, staining with Fe²⁺-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate-containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe²⁺-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 μg of bovine serum albumin, and the limit of detection of this protein is 1 μg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fe²⁺-bathophenanthroline sulfonate is an excellent protein stain.     

The histones of the insect trypanosomatid, Crithidia fasciculata

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.     

Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain

Gel destaining following Coomassie Brilliant Blue (CBB) staining involves the use of toxic reagents. Here we demonstrate the efficacy of various paper adsorbents in adsorbing CBB. Kimwipes adsorbed the best, followed by Teri towels, multifold towels, and Whatman numbers 1 and 3 filter papers. Three Kimwipes completely adsorbed the dye released from a CBB-stained mini-gel. Nonradioactive destain solution can, therefore, be recycled for destaining CBB-stained gels. Stain removal with Kimwipes helps in reducing destain use and in reducing organic liquid waste, and it is 7.5-fold cheaper compared with an available method for CBB disposal. Following this, we determined the suitability of this procedure to remove the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. The dye from a 0.05% CBB staining solution could be removed in 5 to 10 min using 75 Kimwipes. The CBB-adsorbed Kimwipes did not release the stain when squeezed dry even after incubation in various salts over 1 week and in water for 5 weeks. The CBB removed allows its easy disposal as solid waste and will not leach out from solid landfills. Thus, stain removal with Kimwipes helps in disposing CBB in an environmentally friendly manner and allows recycling of destaining solution.     

Colorimetric determination of cell numbers by Janus green staining.

A colorimetric assay using the basic azo dye Janus green has been developed to assess cell numbers in anchorage-dependent cell cultures, with special regard to the enumeration of osteoblastic cells. Therefore, cells are fixed in ethanol and stained with a 0.2% solution of Janus green for 3 min, followed by a destaining step of 1 min in tap water. The addition of diluted hydrochloric acid easily and immediately leads to dye elution from stained cell layers into the acidic supernatant which consequently is transferred into 96-well plates and read on a microplate reader at 595 nm. Working under standardized conditions, Janus green uptake in several cell lines is shown to be linearly correlated with cell numbers over a broad range of cell densities, in MC3T3-E1 cells from about 3% up to more than 300% of confluency. Absolute sensitivity of the assay allows detection of less than 1000 cells/cm(2). In comparison to many other colorimetric assays, the Janus green technique is simple to perform, fast, precise, stable, cheap, and well suited for processing large quantities of samples. Moreover, it is applicable to any culture formate and size, from irregular formed carriers up to 96-multiwell plates.     

Advantages of picrate fixation for staining polypeptides in polyacrylamide gels

When acetic acid-urea polyacrylamide gels with or without Triton X-100 were immersed in 0.1 M Na picrate, pH 7, to which 1/4 vol Coomassie blue staining solution (0.2% in 45% methanol, 10% acetic acid, 45% water) was added, proteins stained rapidly (within a few minutes in gels without Triton and within an hour in gels with Triton) with little or no background staining. Thus protein bands could be observed in a single step with no destaining. The picrate-Coomassie blue method fixed and stained a small peptide (bradykinin, nine amino acids) that was not observed in gels stained with fast green, silver, or Coomassie blue following fixation in 50% trichloroacetic acid. The picrate-Coomassie blue method gave high-contrast bands suitable for densitometry. Gels containing sodium dodecyl sulfate were also stained by the picrate-Coomassie blue method if they were first washed briefly (1 h) in 45% methanol, 10% acetic acid, 45% water, presumably to remove the detergent. These gels also stained rapidly with almost no background.     

Detergent-mediated destaining of coomassie brilliant blue-stained SDS polyacrylamide gels

A simple and one-step detergent-mediated destaining procedure for SDS Polyacrylamide gels for proteins is described. Suspension (5%, w/v) of a commercially available household detergent, Vim Ultra, has been found to be very efficient in destaining polyacrylamide gels without interfering with the resolution of proteins. As compared to the routinely used solvent (methanol-acetic acid-water)-mediated destaining procedure, the present method is economical and user-friendly.     

The use of Coomassie Brilliant Blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels.

A method has been developed to stain rapidly protein zones not only in standard but also in isoelectric focusing polyacrylamide gels. It requires no destaining in either case. The technique makes use of the fact that the G250 form of Coomassie Brilliant Blue exhibits a color change in dilute perchloric acid which is reversed when the dye becomes bound to the protein. Under the conditions used, Ampholine shows little interference. In addition, the method selectively visualizes the arginine-rich histones because of the solubility of the lysine-rich histones in PCA.     

Determination of protein by Coomassie dye-binding in agarose gels

A variation of the Coomassie dye-binding assay for proteins is described. Protein samples were pipetted to the surface of agarose plates in uniformly sized spots and stained with Coomassie Blue G-250. The bound dye was determined by densitometric scanning using double wavelength and flying spot facilities. The response curves were linear in an about 10-fold concentration range with a lower detection limit of 0.5 microgram. No background correction was necessary because unbound dye and most substances known to interfere with other protein assays were removed during the staining and destaining of the agarose gels. Membrane proteins could be analyzed since the samples were applied as solutions in 1% sodium dodecyl sulfate.     

Proteins Specified by Herpes Simplex Virus X. Staining and Radiolabeling Properties of B Capsid and Virion Proteins in Polyacrylamide Gels

ANALYSES OF THE STRUCTURAL PROTEINS OF HERPES SIMPLEX VIRIONS AND OF CAPSIDS CONTAINING VIRAL DNA (B CAPSIDS), AFTER ELECTROPHORESIS IN POLYACRYLAMIDE GELS, REVEALED CONSIDERABLE VARIABILITY IN THEIR PROPERTIES WITH RESPECT TO: (i) retention of Coomassie brilliant blue (CBB) and fast green stains during destaining, (ii) relative optical absorbance of the CBB-protein complex at different wavelengths, (iii) relative efficiency with which (14)C-amino acids are incorporated during early and late periods of the infection cycle, and (iv) capacity to be phosphorylated in vivo. In addition, it was found that protein 22a of B capsids, which does not have an electrophoretically identical counterpart in virions, shares a relatively unique set of staining and radiolabeling properties with virion protein 22, which has a slightly more rapid electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels.     

A Quantitative Procedure for Estimating Cotton Fiber Growth

An improved procedure for quantitation of cotton fiber development, the “stain-destain” method, is reported. Toluidine blue 0 was used to selectively stain fibers subsequently destained in an acid-alcohol solution. Absorbance of the dyecontaining destaining solution was used as a measure of fiber development, and expressed in terms of total fiber units (TFU), one OD unit at 624 nm having been assigned the value of one TFU. Optimum conditions for the procedure, including staining and destaining times and solution to ovule ratios were determined: (1) 20 ovules with associated fibers stained for 15 sec in 80 ml 0.018% toluidine blue O, (2) nonabsorbed dye removed by 60 sec wash, (3) ovuls destajned in 100 ml glacial acetic acid-ethanol-water (10:95:5), (4) absorbance determined after one hr destaining. The procedure is deemed accurate and precise for the purpose intended—quadtation of fiber development as modified by phytohormones or other treatments. Data are shown correlating TFU with fiber length through 14 days postanthesis and an example is given in which the method was used to determine the effect of combined application of gibberellic acid and indoleacetic acid on in vitro cotton fiber development.     

Characterization of the H- and L-subunit ratios of ferritins by sodium dodecyl sulfate-capillary gel electrophoresis

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In contrast, SDS-CGE requires no staining or destaining procedures and the peak quantitation is superior to SDS-PAGE. SDS-CGE is effective in quickly resolving the H- and L-subunits of ferritins from horse spleen, human liver, recombinant human H and L homopolymers, and mixtures of the two- and the single-subunit of a bacterioferritin from Escherichia coli. The technique has also proven useful in assaying the quality of the protein sample from both commercial and recombinant sources. Significant amounts of low-molecular-weight degradation products were detected in all commercial sources of horse spleen ferritin. Most commercial horse spleen ferritins lacked intact H-subunits under denaturing conditions.     

Protein patterns obtained from support-bound gels by combining the images at different stages of destaining

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.     

Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.