Simultaneous Detection of Phosphoproteins and Total Proteins in SDS-PAGE Using Calcon.

A novel fluorescent staining protocol to detect phosphoproteins in sodium dodecyl sulfate-polyacrylamide gels using a fluorescence sensor, 1-(2-hydroxy-1-naphthylazo)-2-naphthol-4-sulfonic acid sodium salt (Calcon), was developed. This method yields results within 135 min, with the sensitivities of 15 ng of α-casein and ß-casein, and 62.5 ng of κ-casein, respectively. Since non-phosphoproteins have shown negative signals that are distinctly different from positive signals of phosphoproteins, this detection method allows one to monitor phosphoproteins with high specificity. Furthermore, a total protein profile can be achieved before a destaining step using a scanner with rapid and low-cost without further total protein staining.

Counterion Dye Staining of Proteins in One- and Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Gel Digestion of Stained Protein for Mass Spectrometry.

A fast and matrix-assisted laser desorption/ionization-mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet to form an ion-pair complex. The protocol including fixing, staining, and quick washing steps can be completed in 1-1.5 h depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Detection of Phosphoproteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Using 8-Quinolinol Stain.

In order to detect phosphoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), an easy and fast fluorescent detection method is described. 8-Quinolinol can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4-8 ng of α-casein and ß-casein, 15-31 ng of ovalbumin and κ-casein within 70 min. The approach utilizing 8-quinolinol could be an alternative staining method for phosphoproteomics.

Sensitive phosphoprotein detection in SDS-PAGE via Anthracene Chrome Red A stain.

Protein phosphorylation, one of the most important post-translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al3+ ) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non-phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA-Al3+ -phosphoprotein complex, unlike non-phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro-Q Diamond stain. ACRA stain can detect 1-2 ng of α-casein and ß-casein, 8-16 ng of ovalbumin (OVA) and κ-casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro-Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8-500 ng, correlation coefficient r = 0.999), α-casein (4-500 ng, r = 0.992), ß-casein (4-500 ng, r = 0.996), and κ-casein (8-500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection.

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.

A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and ß-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research.

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE.

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.

Phosphoprotein staining for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using fluorescent reagent morin hydrate.

A fluorescence-based stain with 3,5,7,2',4'-pentahydroxyflavone (morin hydrate, MH) was designed to stain phosphoproteins in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Al(3+) was applied as a "fixed bridge," providing an efficient energy transfer channel between phosphoprotein and MH, to produce a strong fluorescent complex for the determination of phosphoprotein. As little as 62.5ng of α-casein (7 or 8 phosphates) and ß-casein (5 phosphates), 125ng of ovalbumin (2 phosphates), and κ-casein (1 phosphate) could be visualized with a wide linear dynamic range. In comparison with conventional methods, MH stain is a time-saving method that takes just 90min. It also has good compatibility with routine protein stainings such as Coomassie Brilliant Blue R (CBBR) and SYPRO Ruby for total protein analysis.

Alternative visualization of SDS-PAGE separated phosphoproteins by alizarin red S-aluminum (III)-appended complex.

A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S-aluminum (III)-appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS-PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α- (seven or eight phosphates) and ß-casein (five phosphates), 125 ng of ovalbumin (two phosphates), and κ-casein (one phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125-4000 ng range. As a result, alizarin red S-aluminum (III) stain may provide a new choice for selective, economic, and convenient visualization of phosphoproteins.

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B.

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.

Reverse migration order of sibutramine enantiomers as a function of cyclodextrin concentration in capillary electrophoresis.

The current study demonstrates the reversal of enantiomer migration order (EMO) in capillary electrophoresis (CE) based separations of sibutramines (SIB) as a function of the concentration of two types of cyclodextrin (CD), native ß-CD and acetyl-ß-CD. At normal working concentrations (<10mM) of either CD, (S)-SIB migrated first. However, at CD concentrations greater than 10mM, (R)-SIB was the first to migrate. This study describes factors involved in determining EMO for sibutramine enantiomers at low and high concentrations of CDs. The reversal of EMO could be explained in terms of the opposing effects of the stability and the limiting complex mobility of the SIB-CD complexes. The enantioseparation of SIB with methyl- and 2-hydroxypropyl-ß-CD was possible based on differences in the binding constants of complexes. However, reverse EMO was not observed because of equal mobilities of SIB enantiomers complexed with methyl- and 2-hydroxypropyl-ß-CD.

Promoted negative staining of proteins in SDS-PAGE using Eosin B compounded with magnesium chloride.

In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

Improved conditions for silver-ammonia staining of DNA in polyacrylamide gel.

An improved silver-ammonia staining method for DNA on polyacrylamide gels is described. In this method, staining of DNA using silver-ammonia complex allows high sensitivity, low cost, low toxicity, and simple protocol without requiring fixation and sensitization steps. The protocol takes less than 40 min to complete, with a detection limit of 1.5 pg of single DNA band on polyacrylamide gels, approximately 30-fold higher than that of original silver-ammonia staining method. Furthermore, this novel technique not only exhibits high sensitivity for large DNA fragment, but also shows a better trend to detect low-base-pair DNA compared with other silver staining methods.

A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain.

High-throughput negative detection of SDS-PAGE separated proteins and its application for proteomics.

A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.

GSK-3 phosphorylates delta-catenin and negatively regulates its stability via ubiquitination/proteosome-mediated proteolysis.

Delta-catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby delta-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates delta-catenin and thus affects its stability. Initially, we found that the level of delta-catenin was greater and the half-life of delta-catenin was longer in GSK-3beta(-/-) fibroblasts than those in GSK-3beta(+/+) fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3alpha and -3beta kinase dead constructs, consistently showed that the levels of endogenous delta-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3alpha and -3beta interact with and phosphorylate delta-catenin. The phosphorylation of DeltaC207-delta-catenin (lacking 207 C-terminal residues) and T1078A delta-catenin by GSK-3 was noticeably reduced compared with that of wild type delta-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr(1078) residue of delta-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased delta-catenin levels and caused an accumulation of ubiquitinated delta-catenin. It was also found that GSK-3 triggers the ubiquitination of delta-catenin. These results suggest that GSK-3 interacts with and phosphorylates delta-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

Proteome response to ochratoxin A-induced apoptotic cell death in mouse hippocampal HT22 cells.

Mycotoxins are commonly encountered natural products, and are capable of poisoning animals or humans that inhale mold particles from mycotoxin-contaminated foods. Ochratoxin A (OTA) is produced by Aspergillu ochracus and Penicillium verrucosum, and is often found in cereals and agricultural products. Although previous studies have focused on the potent nephrotoxicity and renal carcinogenicity of OTA, more recent studies suggest that it accumulates in the brain and causes oxidative stress and DNA damage in various brain regions and neuronal populations. In the present study, we undertook to investigate the potential harm caused by environmental exposure to OTA in terms of its effects on neuronal cell viability and proteome profiles. OTA was found to significantly reduce the viabilities of human neuroblastoma SH-SY5Y and mouse hippocampal HT22 cells, as assessed by lactic dehydrogenase release into culture media. Generation of reactive oxygen species was detected in OTA-treated SH-SY5Y and HT22 cells, however, caspase activation and increase in p53 phosphorylation were only detected in HT22 cells, and the expressions of several proteins were found to be significantly altered after treating HT22 cells with OTA. Valosin containing protein, prolyl 4-hydroxylase, Atp5b protein, nucleophosmin 1, eukaryotic translation elongation factor 1 delta isoform, ornithine aminotransferase, prohibitin, and peroxiredoxin 6, which have been suggested to be implicated in the pathogenesis of neurodegenerative disorders, were up-regulated. Our findings suggest that coordinated regulations of molecular networks are involved in the OTA-induced cytotoxicity and that proteome response can be an indicative for neurodegeneration.

Sensitive fluorescent staining for proteomic analysis of proteins in 1-D and 2-D SDS-PAGE and its comparison with SYPRO Ruby by PMF.

A novel fluorescence-based method for protein staining on SDS polyacrylamide gel is described. In this method, proteins are stained using counterion (palmatine and SDS) staining solution, which is inexpensive, easy to perform, and does not involve a destaining step. Fixing and staining of proteins using the counterion protocol take less than an hour. As little as 2 ng of protein can be detected. Another interesting feature of the staining protocol described here is the compatibility with MALDI-TOF MS which shows a similar number of identification score and sequence coverage compared with those of SYPRO Ruby.