Nanotube-grafted polyacrylamide hydrogels for electrophoretic protein separation

Multiwalled carbon nanotube-modified polyacrylamide gels have been employed for the electrophoretic separation of proteins. Two approaches are compared in this investigation, one using nanotubes only as fillers inside the gel matrix and the other using nanotubes as catalyst for polymerization of acrylamide. In both the cases, polymerization of acryl-amide/bisacrylamide has been carried out in situ in the presence of nanotubes dispersed in the gel buffer containing monomer and cross-linker. In the former case, initiator and catalyst have been added after ultrasonication of nanotubes in the gel buffer mixture where the nanotubes play the role of filler. On the other hand, the second approach precludes use of catalyst and involves addition of initiator alone during ultrasonication of nanotubes in the gel buffer containing monomer and cross-linker, which leads to the formation of nanotube-grafted gel after 25 min. When nanotubes are used as a catalyst instead of N,N,N',N'-tetramethylethylenediamine, pore size distribution of the gel matrix and linearity of molecular weight calibration plots are found to be improved. In addition, other issues associated with the use of an external catalyst like handling the moisture-sensitive and corrosive reagent and associated irreproducibility are addressed in this approach.     

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.     

Sample sonication after trichloroacetic acid precipitation increases protein recovery from cultured hippocampal neurons, and improves resolution and reproducibility in two-dimensional gel electrophoresis

Protein precipitation with TCA followed by acetone washing is frequently used to clean samples before 2-DE. However, the difficulty in solubilizing TCA-precipitated proteins causes some variability in 2-D gels and makes it difficult to detect some proteins. In this work we show that sonication of the samples, after TCA precipitation followed by elution in sample buffer, increases total protein recovery, and improves reproducibility and matching ratios between gels when analyzed by specialized software.     

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.     

A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stacking

Acidic PAGE systems using cationic detergents such as benzyldimethyl-n-hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counter stacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line.     

A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV‐LIF (266 nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25 ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214 nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by β‐mercaptoethanol is reported, demonstrating the ease of performing the analysis.     

Improved disc SDS‐PAGE for extraction of low molecular weight proteins from serum

The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS‐PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30 kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS‐PAGE and MALDI‐TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28 kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30 kDa. The experimental results obtained by plate SDS‐PAGE indicated the repeatability was satisfactory.     

Separation and recovery of nucleic acids with improved biological activity by acid‐degradable polyacrylamide gel electrophoresis

One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid‐degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44–84% of DNA. The second conventional (non‐degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid‐degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid‐degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid‐degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.     

脯氨酸硫酯自然化学连接反应中的轨道相互作用

采用密度泛函理论(DFT)方法(M06//B3LYP)对脯氨酸硫酯在自然化学连接(NCL)反应中低反应活性的现象进行了详细的理论研究.通过对脯氨酸和丙氨酸硫酯NCL反应的具体反应路径(Path-Pro和Path-Ala)分别进行计算发现,两个分子的反应路径均先后经历外源硫醇-硫酯交换、硫酯-硫酯交换、分子内S→N酰基化重排三个主要步骤.两者的决速步骤均为第一步骤.其中Path-Pro的总能垒较高,即脯氨酸硫酯的反应活性较低,这与实验结果一致.对两路径的决速步骤进一步考察,发现脯氨酸中αN上的羰基对反应中心羰基的n→π*相互作用使得脯氨酸硫酯的LUMO轨道能量升高,相应羰基C与S(芳基硫醇)原子之间的相互作用较小,从而使得反应能垒升高.     

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 ${\mathrm{PO}}_3^{-}$monomers with toluidine blue or 4′,6-diamidino-2-phenylindole. PolyP chain lengths longer than 62${\mathrm{PO}}_3^{-}$monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich “Type 45”, estimated mean length of 45 ${\mathrm{PO}}_3^{-}$ monomers), trimetaphosphate (trimetaP: 3 ${\mathrm{PO}}_3^{-}$ ring), tripolyphosphate (tripolyP), pyrophosphate (PP_i), and inorganic orthophosphate (o-P_i) were visualized after separation by an in situ hydrolytic degradation process to o-P_i that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol–chloroform extraction was confirmed with the Friedman test. ³¹P diffusion–ordered NMR spectroscopy confirmed that extraction also reduced PP_i diffusivity by <10%. Linear regression between the R_f peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 ${\mathrm{PO}}_3^{-}$monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-P_i, but no PP_i.