Gel Lane Research Articles

Centrifugal Gel Crushing Tips for Gel-Based Proteome Analysis.

We have developed a centrifugal gel-crushing method using a pipet tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipet tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipet tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of prefractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the prefractionated gel slice into optionally small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipet tip. As a result, we observed improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches, including top-down proteomics.

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses.

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.

Changes in the microbiological, physicochemical properties of Chinese traditional fermented Suan rou at ripening fermentation.

This study characterized the changes in the microbiological, physicochemical properties of Suan rou during fermentation via three different techniques (Technique A is a traditional production process. Based on technique A, technique B adds a total of 200 g of sucrose to the thinly sliced meat, and technique C changes the amount of salt in the thinly sliced meat to 200 grams.). Compared to batch A, the samples from batches B and C featured more rapid reduction in pH and generated more TA. Myofibrillar proteins in batches B and C showed higher degradation rate, and several low‐molecular‐weight metabolites were determined on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel lanes. The contents of thiobarbituric acid (TBARS) and total volatile base nitrogen (TVB‐N) and the growth of spoilage bacteria and pathogens were suppressed in the three batches. A relatively compatible acid–salinity proportion was presented in the Suan rou of batches A and B compared with that of batch C. The results show that the Suan rou made by B technology was more palatable acid flavor and abundant nutrition.

Analysis of the Non-Specific Binding Proteins in the RNA Pull-Down Experiment

Background: To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives. Objective: In this study we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Method Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Methods: Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, a comparison between different sample preparation conditions showed that the level of background contaminates was mostly induced by the solid phase support rather than the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins. Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiments.

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

Electrophoretic patterns of proteinuria in feline spontaneous chronic kidney disease.

The purpose of this study was to describe the electrophoretic patterns of proteinuria in cats at risk of and cats with chronic kidney disease (CKD), and to investigate whether the presence of high-molecular-weight (HMW) and low-molecular-weight (LMW) proteins were associated with CKD, proteinuria and/or disease progression. Healthy cats at risk of developing renal disease (n = 17) and cats affected with CKD at different stages (n = 22) were prospectively enrolled and sampled over time. Seventy urine samples were included and assayed with a commercially available sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) method. Each sample (gel lane) was inspected to identify albumin, HMW and LMW proteins, and an electrophoretic pattern (albuminuria, glomerular, tubular, mixed or negative) was assigned accordingly. Fisher's exact test was used to assess the distribution of HMW and LMW proteins in cats grouped according to International Renal Interest Society stage and to the magnitude of proteinuria, and to assess if HMW and LMW proteins at the time of inclusion were associated with the development and progression of CKD. In samples of cats at risk, the most common pattern was glomerular (84.6%); glomerular pattern was also common in cats with CKD (54.2%), although mixed proteinuria and tubular proteinuria were also present (29.5% and 11.4%, respectively). The presence of LMW proteins was associated with CKD (P <0.0001) and to a urine protein:creatinine ratio >0.2 (P = 0.025). Both HMW and LMW proteins were not associated with progression of CKD within 6 months (n = 14). Our results showed that HMW proteinuria is common in healthy cats at risk of developing CKD, although the pathological significance needs to be confirmed. The detection of LMW proteins in urine of cats suspected to be affected by CKD, especially in non-azotaemic, non-proteinuric or borderline proteinuric cats, suggests the presence of kidney damage.

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS

The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained information on individual HBSMC proteins was compared between the two methods, SDS-PAGE is advantageous in visualizing the quantity differences between differently treated samples, whereas nondenatuing 2DE is advantageous in visualizing protein interactions. SDS-PAGE-MS of the supernatant fraction provided the assignment of 2552 proteins and their percent abundance ranged from 3.5% to 2 × 10−4%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10−5%, suggesting that the step of isoelectric focusing served to raise the sensitivity of the method. The proteins in the precipitate fraction, which could not be analyzed by 2DE-MS, were characterized by the abundance of proteins allocated to “membrane” within the category “Cellular component” of UniProtKB database and especially those allocated to “transmembrane” within the subcategory “membrane.” On the other hand, there were about 600 “membrane” proteins which showed more than two-fold higher percent abundance in 2DE-MS than in SDS-PAGE-MS. These results showed that 1D SDS-PAGE and nondenaturing 2DE would provide complementary information on the analysis of proteins and protein interactions in cells.

Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes.

The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein-nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd ) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA-based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. © 2018 by John Wiley & Sons, Inc.

Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis.

Hereditary hemochromatosis (HH) is characterized by excessive iron absorption in the intestine, which can lead to failure of vital organs such as the heart, liver, and pancreas. Among northern Europeans, HH is most often associated with the C282Y and H63D mutations of the HFE gene. We developed a test that allows screening for both mutations in a single reaction. A multiplex allele-specific PCR was developed for simultaneous genotyping of the H63D and C282Y HFE mutations. PCR fragments were designed such that the resulting PCR product can be analyzed in a single polyacrylamide gel lane. Test results from our multiplex assay were concordant with genotypes of 55 Canadian patients with suspected hemochromatosis, which had previously been established by allele-specific PCRs that targeted H63D and C282Y in separate reactions. Molecular diagnostic detection of H63D and C282Y mutations can be achieved by a variety of methods, but these are not necessarily time-efficient or economical. Multiplex allele-specific PCR is an excellent tool for molecular diagnostic screening for H63D and C282Y mutations in patients with suspected hemochromatosis. This method is inexpensive, accurate, and highly efficient in terms of labor, throughput, and turnaround time.

A method to quantify genotoxicity of malathion in rainbow trout using the weighted averaging

DNA breakage has been frequently used as a biomarker of the pesticide toxicity. The present study introduced a method to quantify the DNA breakage in Oncorhynchus mykiss exposed to the pesticide malathion. Specimens were exposed to different concentrations of malathion for 1–9 days and their gill and liver were sampled. DNA was extracted and electrophoresed using agarose gel. The pixel density curves were obtained from the gel smears. The area under the curves was arbitrarily divided from three up to seven segments using a Java macro in the software ImageJ. Some weighted averaging methods were used to calculate DNA breakage in each gel lane. Akaike information criterion (AIC) was used to find the best analysis of variance. The liver was more sensitive than the gill showing a larger number of significant differences among the specimens exposed to various concentrations of malathion. The geometric weighted averaging on the data extracted from the seven-segment pixel density curve resulted to the lowest AIC. The double-strand DNA breakage of O. mykiss was able to detect malathion in freshwater in concentrations over 0.05 mg L−1.

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

Fabrication of an Open Microfluidic Device for Immunoblotting.

Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an "enclosed" microchannel for PAGE and blotting to an "open" PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

Correction.

The Plant JournalVolume 91, Issue 6 p. 1129-1130 CorrectionFree Access Correction This article corrects the following: Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes Naïra Naouar, Klaas Vandepoele, Tim Lammens, Tineke Casneuf, Georg Zeller, Paul Van Hummelen, Detlef Weigel, Gunnar Rätsch, Dirk Inzé, Martin Kuiper, Lieven De Veylder, Marnik Vuylsteke, Volume 57Issue 1The Plant Journal pages: 184-194 First Published online: September 19, 2008 First published: 01 September 2017 https://doi.org/10.1111/tpj.13653AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In the article “Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes” by Naïra Naouar et al. (2009), Figure 7 (original), 7 (corrected) holds duplicated and incorrect joined gel lane fragments. Because only part of the original data could be retrieved, the experiments were reproduced according to today's experimental standards, using quantitative RT-PCR instead of RT-PCR. As the original E2Fa serum lost its original quality, a new serum was used (kindly provided by Dr. Magyar Zoltan) of which the details will be described elsewhere. E2Fa and E2Fb ChIP experiments were performed on an E2Fa overexpressing and, wild type Col-0 line respectively. The data confirm that the majority of the predicted E2F target genes are bound by E2Fa or E2Fb, supporting the previous statement that the Affymetrix Tilling 1.0R array allowed prediction of novel E2F target genes. The third author Tim Lammens takes the entire responsibility for making the faulty collage and for not informing the other authors. Co-corresponding authors Lieven De Veylder and Marnik Vuysteke take responsibility for not recognizing the faulty collage before submitting the paper for publication. All raw data and materials described are freely available upon request. Figure 7 (original)Open in figure viewerPowerPoint Chromatin immunoprecipitation (ChIP) analysis confirming direct E2Fa/E2Fb binding to the promoter of 10 randomly selected potential direct targets (gene identifiers on the left) of E2Fa-DPa, identified uniquely on the Tiling 1.0R platform. ChIP was performed on 8-day-old E2Fa-DPaOE seedlings using E2Fa- or E2Fb- specific antibodies. An immunoprecipitation without any added antibody served as a control. Figure 7 (corrected)Open in figure viewerPowerPoint Chromatin immunoprecipitation (ChIP) analysis confirming direct E2Fa/E2Fb binding to the promoter of 10 randomly selected potential direct targets (gene identifiers on the left) of E2Fa-DPa, identified uniquely on the Tiling 1.0R platform. ChIP was performed using E2Fa- (E2Fa IP) or E2Fb-specific (E2Fb IP) antibodies on 8-day-old E2FaOE and wild type Col-0 seedlings, respectively. An immunoprecipitation without any added antibody served as a control (No Ab). The ACT2 (At3g46520) and CDKA;1 (At3g48750) genes serve as negative controls, whereas UVI4 (At2g42260) represents a positive control. The dashed lines indicate the background level. Reference Naouar, N., Vandepoele, K., Lammens, T. et al. (2009) Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes. Plant J. 57, 184– 194. Volume91, Issue6September 2017Pages 1129-1130 FiguresReferencesRelatedInformation

The mitochondrial complexome of Arabidopsis thaliana.

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein-protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot-gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re-annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource (https://complexomemap.de/at_mito_leaves) to deposit the data and to allow straightforward access and custom data analyses.

Determining Protease Substrates Within a Complex Protein Background Using the PROtein TOpography and Migration Analysis Platform (PROTOMAP).

The PROtein TOpography and Migration Analysis Platform (PROTOMAP) approach is a degradomics technique used to determine protease substrates within complex protein backgrounds. The method involves protein separation according to protein relative mobility, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel lanes are then sliced into horizontal sections, and in-gel trypsin digestion performed for each gel slice. Extracted peptides and corresponding proteins are identified using liquid chromatography-tandem mass spectrometry and bioinformatics. Results are compiled in silico to generate a peptograph for every identified protein, being a pictorial representation of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins shown by their peptograph to have migrated further through the gel (i.e., to a lower gel slice) in the lane containing the active protease(s) of interest, as compared to the control, are deemed putative protease substrates. PROTOMAP has broad applicability to a range of experimental conditions and protein pools. Coupling this with its simple and robust methodology, the PROTOMAP approach has emerged as a valuable tool with which to determine protease substrates in complex systems.

Analysis on DNA methylation of mosaic and moxa type leaves of Aconitum carmichaeli by MSAP

An MSAP analysis method was established for detecting DNA methylation of Aconitum carmichaeli leaves, and the DNA methylation of different leaf shapes and different leaf position was analyzed by MSAP. The study made experiments on the leaves of different position of mosaic and moxa leaf type A. carmichaeli, researched the effects of restriction digestion of genomic DNA by using two restriction enzymes, screened the suitable selective amplification primers, and analyzed the methylation differences of leaves by calculating the 6% acrylamide gel electrophoresis bands and lane. The best reaction system of MSAP was obtained, under the conditions of 37 ℃, the 16 h incubated time was more suitable for 150 ng DNA, and 25 pairs of selective amplification primers were selected from 256 pairs. Totally, 273 electrophoresis bands were obtained by 25 pairs of selective primers, including 228 non methylation or single chain methylation bands,27 double chain methylation bands,and 18 single stranded methylation bands, the total methylation rate was 16.48%. The methylation rate was slightly different in mosaic and moxa leaf type A. carmichaeli leaf, which were 15.36%, 14.34%, respectively, and article 8, article 6 nucleotide fragments of genome methylation modification differences were obtained, accounted for 3%, 2.26% of the total number of bands. Based on this study it can provide new ideas for molecular identification, breeding and cultivation, and genetic evolution of A. carmichaeli.

Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel.

High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The kon is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify koff, immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine kon and koff for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines kon and koff in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.

Estimating the DNA strand breakage using a fuzzy inference system and agarose gel electrophoresis, a case study with toothed carp Aphanius sophiae exposed to cypermethrin.

The DNA breakage has been widely used in ecotoxicological studies to investigate effects of pesticides in fishes. The present study used a fuzzy inference system to quantify the breakage of DNA double strand in Aphanius sophiae exposed to the cypermethrin. The specimens were adapted to different temperatures and salinity for 14days and then exposed to cypermethrin. DNA of each specimens were extracted, electrophoresed and photographed. A fuzzy system with three input variables and 27 rules were defined. The pixel value curve of DNA on each gel lane was obtained using ImageJ. The DNA breakage was quantified using the pixel value curve and fuzzy system. The defuzzified values were analyzed using a three-way analysis of variance. Cypermethrin had significant effects on DNA breakage. Fuzzy inference systems can be used as a tool to quantify the breakage of double strand DNA. DNA double strand of the gill of A. sophiae is sensitive enough to be used to detect cypermethrin in surface waters in concentrations much lower than those reported in previous studies.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

One-dimensional proteomic profiling of Danio rerio embryo vitellogenin to estimate quantum dot toxicity.

BackgroundVitellogenin (Vtg) is the major egg yolk protein (YP) in most oviparous species and may be useful as an indicator in ecotoxicological testing at the biochemical level. In this study, we obtained detailed information about the Vtgs of Danio rerio embryos by cutting SDS-PAGE gel lanes into thin slices, and analyzing them slice-by-slice with (MALDI-TOF) mass spectrometry.ResultsWe conducted three proteomic analyses, comparing embryonic Danio rerio Vtg cleavage products after exposure for 48 h to CdSecore/ZnSshell quantum dots (QDs), after exposure to a mixture of the components used for quantum dot synthesis (MCS-QDs), and in untreated embryos. The Vtg mass spectrometric profiles of the QDs-treated embryos differed from those of the unexposed or MCS-QDs-treated embryos.ConclusionThis study demonstrates the possible utility of Vtg profiling in D. rerio embryos as a sensitive diagnostic tool to estimate nanoparticle toxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0072-7) contains supplementary material, which is available to authorized users.