Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

During SDS‐PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ～110 residues‐long, sequence‐homologous, structurally‐analogous, extracellular domains of human E‐ and N‐cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively‐charged and positively‐charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater‐than‐expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS‐protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.     

血清中低分子量蛋白质的提取和鉴定

采用改进的圆盘凝胶电泳提取人血清中低分子量蛋白质, 去除了血清中分子量大于3×104的蛋白质, 将提取的低分子量蛋白质热变性后直接在溶液中酶解成肽, 经液相色谱-质谱分析, 并进行Mascot数据库检索, 确认出人血清中97种蛋白质.     

Gel Chromatographic Fractionation and Partial Physicochemical Characterization of Gelatins

Summary Gel chromatography on Sephacryl S-400 HR was used to fractionate a type A and a type B gelatin on a semi-preparative scale. Fractionation was performed at 40 °C in 50 mM ammonium acetate, 0.02% (w/v) sodium azide. Five fractions, enriched in microgels, -chains, -chains, -chains and subfragments, were isolated. Their molar mass distribution was controlled by FPLC on Superose 6 and SDS-PAGE. No significant differences in amino-acid composition and hexose content were observed between the original gelatins and their fractions. The specific absorption coefficient at 230 nm was found to be the same for both type A and type B gelatin and the chromatographic fractions (2.0 L·g^–1·cm^–1).Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Characterization of carbon nanotubes and analytical methods for their determination in environmental and biological samples: A review

In the present paper, a critical overview of the most commonly used techniques for the characterization and the determination of carbon nanotubes (CNTs) is given on the basis of 170 references (2000–2014). The analytical techniques used for CNT characterization (including microscopic and diffraction, spectroscopic, thermal and separation techniques) are classified, described, and illustrated with applied examples. Furthermore, the performance of sampling procedures as well as the available methods for the determination of CNTs in real biological and environmental samples are reviewed and discussed according to their analytical characteristics. In addition, future trends and perspectives in this field of work are critically presented.     

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 β‐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), β‐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.     

SDS disc electrophoresis of proteins in homogeneous, low-concentrated polyacrylamide gels

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.     

Reversed-phase high-performance liquid chromatographic, size exclusion chromatographic and polyacrylamide gel electrophoretic studies of glycinin: evidence for molecular species and their association-dissociation

Isolation and purification of glycinin and its molecular species from an Indian soybean variety (JS-335) was achieved using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Glycinin was found to have two molecular species (glycinin I and II), and only glycinin I underwent reversible dissociation-association system into alpha and beta species. Glycinin I and II were not found to constitute a dissociation-association system. Glycinin II also did not dissociate under varying conditions of time, pH and ionic strength of buffer. Various species so dissociated were isolated, purified and characterized.     

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.     

In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases

Based on digital image analysis techniques and a series of optimizations in native electrophoresis, a new direct method to simultaneously detect the intrinsic properties of each active component in the enzymatic system of glycoside hydrolase was established. The key technique is that the concentration changes of substrate (or product) on the gel can be determined quantitatively by the gray value changes of the corresponding band after electrophoretic separation. In this manner, the catalytic characteristics of each glycoside hydrolase component were demonstrated in situ and were easily determined after immersing the gel in a series of solutions containing substrates or their derivatives. Because of its high throughput, great sensitivity, and convenient operation, this method can be used to demonstrate the natural diversity of glycoside hydrolases and to study spatial and temporal regulation in multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of glycoside hydrolases.     

X-ray absorption fine structure of artificial antigens for cadmium

Immunoassay technology as a quick and large-scale screening method to detect metal ions in foods and environmental samples has rapidly been developed due to several advantages over conventional instrument-intensive methods. Unlike biomacromolecule, metal ions are haptens without immunogenicity, so successful preparation of artificial antigens is the first critical step for establishing immunoassay methods for them. In the current paper, cadmium ions were conjugated to BSA and OVA, respectively, using bifunctional chelator, p-SCN-Bn-DTPA. The ultraviolet analysis indicated that the maximum absorption peak of Cd–p-SCN-DTPA–BSA and Cd–p-SCN-DTPA–OVA had a small peak shift and an apparent absorbance increase compared to that of BSA and OVA, and the extents of substitution of ?-amino in both conjugates were 51.2% and 58.6%, respectively. In addition, the EXAFS of conjugates implied that Cd²⁺ coordinated with N and O atoms of DTPA in artificial antigens, the coordination type and number of Cd–DTPA, Cd–p-SCN-Bn-DTPA–BSA, Cd–p-SCN-Bn-DTPA–OVA were the same. XANES region and geometries of the three compounds were also same. These results implied that the three antigens had the similar local structure and atomic geometry.This was the first time that the XAFS was attempted for the identification of artificial heavy metal ion antigens.