Polyacrylamide gel electrophoresis (PAGE) is routinely used for electrophoretic characterization of biomolecules. In this study we demonstrate that this technique is quite useful for the characterization of small molecules, however, it becomes inadequate for the resolution of high‐molecular‐weight macromolecules like MUC2 mucins. We show that agarose gel electrophoresis (AGE) constitutes a better medium through which MUC2 macromolecules can penetrate and hence be resolved. MUC2 molecules obtained from PC/AA (a benign colonic adenoma cell line) were extracted, and their molecular weights (varying between 15–32 × 106 Da) determined using Laser Light Scattering (LLS). These mucin molecules were reduced until 5–6 × 106 subunits were obtained. The reduced and unreduced mucin molecules were run on an AGE and Western blotting and visualized, but were too large for PAGE. For this reason, the mucin subunits were treated with trypsin to obtain 500–600 kDa fragments. These tryptic fragments were then run on PAGE and AGE, and their resolutions compared. We conclude that AGE is a more useful electrophoretic method for characterization of macromolecules like mucin glycoproteins than PAGE.
Agarose gel electrophoresis of PC/AA cell layer extract (U: unreduced, intact form, R: reduced form). 相似文献
Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions. 相似文献
In the present work, we introduce a new type of DNA variation detection. This method represents a transfer of melting gel technique onto multicapillary electrophoresis DNA sequencing instrument with further improvements to achieve maximum sample throughput while maintaining a high performance. The main improvement comes from application of cycling (revolving) temporal temperature gradient in place of a single-sweep gradient, commonly used in similar gel-based techniques. This improvement enables utilization of multiple-injection technique, in which multiple samples are injected into the same capillary (or sets of capillaries) separated by predefined time intervals of partial electrophoresis. The periodic oscillation of the temperature results in identical separation conditions of all samples injected in such series. Using this novel approach, we demonstrate a dramatic increase in separation throughput by turning a standard commercial 96-capillary array instrument into a semicontinuous flow mutation detection system capable to screen over 15 000 samples in 24 h of operation on a single 96-capillary commercial instrument. This represents a 10-fold increase in sample throughput over the current comparable technology. 相似文献
Many proteins contain iron as metal ion either within their own structures or bound to their active sites. These iron-containing proteins are involved in numerous biological processes and some of them serve as biomarkers of clinical pathologies, not only related to iron homeostasis but also to other physiological disorders. Thus, a variety of analytical strategies have been developed over the last years in order to conduct studies on Fe-containing proteins. Among them, mass spectrometric (MS) methods still remain as preferred tools since they provide the capabilities of structure elucidation together with quantitative possibilities. Therefore, in this work we have tried to summarize the most recent applications of elemental and molecular mass spectrometric-based methods for the characterization (mostly qualitative but quantitative in some cases) of the high abundant Fe-containing proteins used for clinical diagnosis. 相似文献
Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin‐containing poly‐horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP‐secondary antibody instead of HRP‐secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin‐conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin‐conjugated PolyHRP resulted in as much as a 110‐fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin‐conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin‐conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future. 相似文献
Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products. 相似文献
A purification strategy for rapidly obtaining recombinant human thyrotropin (rhTSH) was designed based on size exclusion and reversed-phase high-performance liquid chromatographic (HPLC) analysis, carried out on hTSH-secreting CHO cell conditioned medium. These analyses permitted the identification of the main contaminants to be eliminated. Considering that hTSH is highly hydrophobic and elutes only with the addition of organic solvents, hydrophobic interaction chromatography was adopted as the first purification step; this resulted in the elimination of, among others, the major contaminant. A second purification step, based on size exclusion chromatography, was then utilized, being effective in the elimination of other previously identified contaminating proteins. Useful purity, as high as 99% at the chemical reagent level, and recoveries (37%) were obtained by adopting this two step strategy, which also provided adequate material for physico-chemical, immunological and biological characterization. This included matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS), Western blotting analysis, in vivo biological assay, size-exclusion HPLC (HPSEC) and reversed-phase HPLC (RP-HPLC) analysis, which confirmed the integrity and bioactivity of our rhTSH in comparison with the only two reference preparations available at the milligram level of native (hTSH-NIDDK) and recombinant (Thyrogen) hTSH. Thyrogen and rhTSH-IPEN, when compared to pit-hTSH-NIDDK, presented more than twice as much biological activity and about 7% increased molecular mass by MALDI-TOF-MS analysis, an accurate heterodimer mass determination providing the Mr values of 29,611, 29,839 and 27,829, respectively. The increased molecular mass of the two recombinant preparations was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPSEC analysis. Comparing the two recombinant preparations, minor though interesting physico-chemical and biological differences were also observed. 相似文献
The synthesis and characterization of a series of polyammonium β-alanine containing dendrimers 4-6 were reported. Such polycationic species were shown to exhibit electrophoretic behavior similar to that of protein molecules such as lysozyme and aprotinin in acidic polyacrylamide gel electrophoresis. It was also found that their electromobility was directly related to their molecular size, and hence such biomimetic dendrimers are potential molecular weight markers for protein analysis. 相似文献