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Microanalysis of SDS-PAGE electroblotted proteins   总被引:5,自引:0,他引:5  
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Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.  相似文献   

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Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.  相似文献   

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Biological networks are powerful tools for predicting undocumented relationships between molecules. The underlying principle is that existing interactions between molecules can be used to predict new interactions. Here we use this principle to suggest new protein-chemical interactions via the network derived from three-dimensional structures. For pairs of proteins sharing a common ligand, we use protein and chemical superimpositions combined with fast structural compatibility screens to predict whether additional compounds bound by one protein would bind the other. The method reproduces 84% of complexes in a benchmark, and we make many predictions that would not be possible using conventional modeling techniques. Within 19,578 novel predicted interactions are 7,793 involving 718 drugs, including filaminast, coumarin, alitretonin and erlotinib. The growth rate of confident predictions is twice that of experimental complexes, meaning that a complete structural drug-protein repertoire will be available at least ten years earlier than by X-ray and NMR techniques alone.  相似文献   

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A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.  相似文献   

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Graphitic nanofibres (GNFs), 100–200 nm in diameter and 5–20 μm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 μg ml−1 for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol μl−1 (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.  相似文献   

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A hydrophylic isothiocyanate resin has been synthesized starting from a commercial polyacrylic acid. Lysine-containing proteins easily react with this support in aqueous media, yielding an insoluble derivative able to be sequenced by conventional methods.  相似文献   

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The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.  相似文献   

10.
The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.  相似文献   

11.
Summary Porous glass fiber has a very high surface area and good mechanical properties that make it an excellent support for biocatalyst immobilization. By packing aligned glass fibers in a tubular reactor such that the fibers are all parallel to the axis of the tube, the resulting pressure drop is considerably smaller than for a similar bed of packed beads. The utility of this support was demonstrated by immobilizing -glucoamylase by silane-glutaraldehyde coupling, and measuring its activity toward converting maltose to glucose. Using optimized immobilization conditions, an enzyme loading of 1.5 mg protein perm 2 surface area was obtained, with an activity of 370 units/g glass at 50°C. The half-life of the immobilized glucoamylase was more than twice as long as that of the free enzyme.  相似文献   

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A method for determination of amino acid composition of proteins separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes is described. A single blotted band containing 50 to 200 pmoles of protein was cut out and submitted to acid hydrolysis with HCl followed by derivatization with phenylisothiocyanate. The amino acid derivatives were separated by reverse phase high-performance liquid chromatography. Bovine serum albumin, lysozyme, myoglobin, ovalbumin, soybean trypsin inhibitor and carbonic anhydrase were analyzed; the results revealed a good correspondence with reported values. This can be considered an analytical method to determine the amino acid composition of samples from microquantities of protein mixtures, particularly in those cases in which SDS-polyacrylamide gel electrophoresis is the most suitable separation system.  相似文献   

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The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.  相似文献   

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There are numerous viral proteins known to date to modulate protective responses of their hosts. Representatives of the Poxviridae family have the greatest number of genes coding for proteins, inhibiting inflammatory responses, activities of interferons, regulating immune reactions and other protective mechanisms of macroorganisms, among viruses. This review regards poxviral immunomodulatory proteins--namely, complement-binding proteins, inhibitors of serine proteases, chemokine- and TNF-binding proteins --that were shown to be efficient therapeutics in various animal models of inflammatory and autoimmune diseases. The prospects of their usage in clinical practice for treating human inflammatory and autoimmune disorders are discussed.  相似文献   

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In this report we describe the use of a novel, experimental, polyvinylidene fluoride-based membrane with a cationic surface for the isolation by electroblotting of small amounts of proteins separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis. The membrane is characterized by a surface that mediates primarily ionic protein/membrane interactions and that allows the recovery of adsorbed proteins at high yields under relatively mild conditions. In electroblotting experiments, the novel membrane has a binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted proteins in situ. Intact electroblotted proteins, or fragments thereof, were eluted at high yields. Further structural analysis is demonstrated using reverse-phase high-performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed-liquid- or solid-phase automated sequence analysis.  相似文献   

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