Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.     

Application of Neuhoff's optimized Coomassie brilliant blue G-250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.     

Fast isoelectric focusing of milk proteins on small ultrathin polyacrylamide gels containing urea

The preparation of 0.45 mm thin polyacrylamide gels, containing urea, for horizontal micro isoelectric focusing of milk proteins with PhastSystem is described. Isoelectric focusing in the small gels, stained either with Coomassie Brilliant Blue R-250 or with the more sensitive silver stain, affords a fast and sensitive procedure for an analysis of milk and cheese proteins. The procedure can be effectively exploited in detecting adulteration in ovine cheese with bovine milk.     

Gel electrophoretic analysis of chitosan hydrolysis products.

Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5% v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R-250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10% w/v polyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33% w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R-250 was found to be better than Calcofluor White M2R for staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system. To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.     

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Interference of the carbohydrate moiety in coomassie brilliant blue R-250 protein staining

The binding of Coomassie Brilliant Blue R-250 to several species of bovine pancreatic ribonuclease is affected by the presence of a carbohydrate moiety in the enzyme molecule. Enzymic deglycosylation of several chromatographic fractions of ribonuclease, which have different degrees of glycosylation, results in increased staining by Coomassie Brilliant Blue R-250. Ovalbumin and other glycoproteins tested show similar behavior. The results indicate that carbohydrate moieties may represent a common hindrance to the binding of Coomassie Brilliant Blue dyes to glycoproteins.     

A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.

A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.     

Using coomassie blue to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels.

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R-250 staining and destaining to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels, prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.     

Improved detection of human pancreatic proteins separated by two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis using a combined staining procedure.

In order to assess secretory pancreatic proteins in a two-dimensional isoelectric focusing/sodium dodecyl sulphate electrophoresis gel, a highly sensitive double-staining method with Coomassie Brilliant Blue followed by silver stain was used. This combined procedure afforded more distinct spots and additional bands, particularly glycoproteins, than either silver or Coomassie Blue staining alone. As measurements of dye volumes by densitometry have shown, double staining of two-dimensional separated pancreatic proteins is up to twenty times more sensitive than the usual Coomassie Brilliant Blue staining.     

2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: application to the analysis of IgG

A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies.     

Comparison of fluorescent stains: relative photostability and differential staining of proteins in two-dimensional gels

The fluorescence of proteins stained with Deep Purple and SYPRO Ruby was measured over a time course of UV transillumination to determine the relative photostability of each stain. Mean spot fluorescence (n = 200 matched spots) in gels stained with Deep Purple decreased 27% following 2 min of UV transillumination, compared to SYPRO Ruby, which decreased 17%. After 19 min, an 83% decrease in Deep Purple fluorescence was observed, compared to 44% for SYPRO Ruby. By interpolation, the half-life of Deep Purple fluorescence was estimated to be approximately 6 min. The half-life of SYPRO Ruby fluorescence was not reached during the 19 min time course. Further, differential staining of proteins was observed in gels stained with Deep Purple and SYPRO Ruby as compared to colloidal Coomassie Brilliant Blue and silver staining.     

Suitability of staining techniques for the detection and quantitation of nonhistone high mobility group proteins.

Three different staining techniques were compared for the detection of nonhistone high mobility group (HMG) proteins after acidic urea-polyacrylamide gel electrophoresis. Silver staining after glutaraldehyde fixation provides the highest detection sensitivity. Because of the acid solubility of HMG proteins special care has to be taken concerning fixation. Staining with colloidal CBB G-250 according to Neuhoff et al. is superior in sensitivity and reliability of quantitation when compared with noncolloidal Coomassie Brilliant Blue R-250. High detection sensitivity and reproducibility of quantitation are prerequisites for studying the tissue-specific expression of HMG proteins. In the present study tissue-specific differences in the molar amounts of various HMG proteins in thymus and erythrocytes of the chicken are documented by application of the methods tested.     

Analysis of the genetic polymorphism of coagulation factor XIIIB (FXIIIB) by isoelectric focusing

The genetic variants of the coagulation factor XIIIB (FXIIIB) were analyzed by isoelectric focusing, carried out in agarose gels and followed by immunofixation. The FXIIIB phenotypes were visualized by a combined staining procedure with Coomassie Brilliant Blue R-250 and silver nitrate. Improved resolution was accomplished in polyacrylamide gels by hybrid isoelectric focusing in immobilized pH gradients supplemented with carrier ampholytes. We examined a total of 1,604 unrelated, healthy individuals from Southern Germany. The frequencies for the FXIIIB alleles were B*1 = 0.7581, B*2 = 0.0843, B*3 = 0.1568 and B*4 = 0.0019. The theoretical exclusion rate for disputed paternity is 22.35%.     

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.     

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.     

Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.