Jung-Kap Choi

Direct Blue 71 staining of proteins bound to blotting membranes

A sensitive staining method for protein blots using Direct Blue 71 is described. It is based on the selective binding of dye molecules to proteins in acidic solution and produces bluish violet colored bands. It is a simple and rapid procedure, involving only staining and rinsing steps that occur within 7 min. The sensitivity of this method is 5—10 ng of protein on nitrocellulose (NC) and 10—20 ng on polyvinylidene difluoride (PVDF), which is tenfold better than that of the commonly used Ponceau S staining. Moreover, the staining is reversible for subsequent immunostaining, without impairing immunoreactivity. To remove the dye from the developed bands, changes in pH and hydrophobicity of the solvent are required. Due to its sensitivity, rapidity, simplicity, and low cost, this stain may be more practical than other dye‐based stains or metal‐based stains for routine laboratory purposes.

Counterion‐dye staining method for DNA in agarose gels using crystal violet and methyl orange

Sensitive and safe methods for visualization of DNA in agarose gels are described. 0.001% crystal violet dissolved in distilled water was used for DNA staining on agarose gels and it could detect as little as 16 ng of DNA (3 kb, pGem‐7Zf/EcoRI) without destaining procedure. The detection limit is four times lower than that of ethidium bromide. To improve the sensitivity, we studied a counterion‐dye staining method using methyl orange as a counterion‐dye which contributes to reduce excessive background staining by crystal violet. Dye concentration, pH of staining solution, mixing molar ratio of two dyes, and staining times were optimized for the counterion‐dye staining. By the staining with a mixed solution of 0.0025% crystal violet and 0.0005% methyl orange in distilled water, 8 ng of the 3 kb DNA in an agarose gel was detected within 30 min.

Isoorientin induces Nrf2 pathway-driven antioxidant response through phosphatidylinositol 3-kinase signaling.

Because oxidative stress is involved in the pathogenesis of various chronic diseases and the aging process, antioxidants that can increase the intrinsic antioxidant potency are proposed as desirable therapeutic agents to counteract oxidative stress-related diseases. NF-E2-related factor-2 (Nrf2) is a transcription factor that regulates important antioxidant and phase II detoxification genes, and therefore, the molecule that regulates nuclear translocation of Nrf2 and the induction of antioxidative proteins is thought to be a promising candidate as a cytoprotective agent for oxidative stress. In the present study, we show that isoorientin (Iuteolin 6-C-β-D-glu-coside) obtained from the leaves ofSasa borealis upregulates and activates Nrf2, and has protective ability against oxidative damage caused by reactive oxygen intermediates in HepG2 cells. Isoorientin induces increase in the level of antioxidant enzyme proteins, especially NQO1, and the cytoprotective and antioxidative effects of isoorientin are PI3K/Akt pathway-dependent. Together with direct radical scavenging activity, the novel effect of isoorientin on the regulation of antioxidative gene expression provides attractive strategy to prevent diseases associated with oxidative stress and attenuate the progress of the diseases.

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.

Precise manipulation of a microrobot in the pulsatile flow of human blood vessels using magnetic navigation system

This paper proposes a method to precisely manipulate a microrobot in the pulsatile flow that simulates the flow characteristics of human blood vessels by utilizing the electromagnetic transfer function of a magnetic navigation system (MNS). The frequency response characteristics of the MNS were utilized so that the input voltages in each coil can precisely generate the required time-varying magnetic force of a microrobot. An experiment which successfully anchoring a microrobot in a pulsatile flow was conducted to verify the proposed method.

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.

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 1h. 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 1h with a detection limit of 0.2 ng/band.

Fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counter ion-dyes, coomassie brilliant blue R-250 and neutral red

A fast and sensitive protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using both an acidic dye, Coomassie Brilliant Blue R-250 (CBBR) and a basic dye, Neutral Red (NR) is described. It is based on a counter ion-dye staining technique that employs oppositely charged two dyes to form an ion-pair complex. The selective binding of the free dye molecules to proteins in an acidic solution enhances the staining effect of CBBR on protein bands, and also reduces gel background. It is a rapid staining procedure, involving fixing and staining steps with short destaining that are completed in about 1 h. As the result, it showed two to fourfold increase in sensitivity comparing with CBBR staining. The stained protein bands can be visualized at the same time of staining.

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 β‐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.

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.