Determination of molecular weight of native proteins by polyacrylamide gradient gel electrophoresis

An improved method for the estimation of molecular weights of native proteins by polyacrylamide gel electrophoresis, in 9 cm x 9 cm x 0.05 mm 4-20% T fabric reinforced gradient gels, is described. Plotting the logarithm of the relative mobilities of proteins versus gel concentrations produces lines whose slopes are related to molecular weights.     

Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (相似文献   

4:3-β-naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester as a fluorescent labeling reagent for amino acids and oligopeptides in high-performance liquid chromatography

Summary 4:3-β-Naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester (NPA-OSu) is a highly sensitive and moderately reactive derivatizing reagent with a naphthapyrone moiety as fluorophore and anN-hydroxysuccinimidyl active ester as reactive group toward amino compounds. It is readily prepared in two steps. The fluorescence properties of NPA-OSu and its hydrolysis product have been studied in detail, and the conditions for derivatization and separation of the NPA-OSu derivatives of some amino acids and oligopeptides have been investigated. Atλ _ex = 352 nm andλ _em = 422 nm the detection limits (signal-to-noise ratio=3) for amino acids and oligopeptides reached fmol levels, for injection of 20 μL; this sensitivity was comparable with that obtained by use of 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester as derivatizing reagent in the analysis of amino acids by capillary electrophoresis with laser-induced-fluorescence detection.     

Purfication of synthetic cardiotoxin by affinity chromatography

A polypeptide containing 60 amino acids with 4 disulphide bonds, synthesized by the solid-phase method, was highly purifed by anticardiotoxin-Sepharose affinity chromatography following gel filtration and CM-cellulose chromatography. The identification of the final product as cardiotoxin was confirmed by thin-layer chromatography on silica gel, polyacrylamide gel electrophoresis, amino acid analysis, circular dichroism spectra, N-terminal analysis and four biological tests.     

N-hydroxysuccinimidyl-fluorescein-O-acetate for precolumn fluorescence derivatization of amino acids and oligopeptides in liquid chromatography

A new fluorescent derivatizing reagent, N-hydroxysuccinimidyl-fluorescein-O-acetate, is used for the high-performance liquid chromatographic analysis of amino acids and oligopeptides. This reagent has the advantages of high-detection sensitivity in the visible region, specifically with amino groups, mild derivatization conditions, and little interference induced. The fluorescence properties of the reagent and its derivatives with amino acids and oligopeptides are studied. The conditions of the derivatization are investigated in detail. In the mobile phase of methanol-water (42:58, v/v) containing a 10 mM pH 5.0 citric acid-Na2HPO4 buffer, six amino acids and oligopeptides are separated in 20 min with fluorescence detection at excitation and emission wavelengths of 492 and 513 nm, respectively, with the detection limits for injected standards ranging from 0.64 to 12 fmol.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

Nafion membrane electrophoresis with direct and simplified end-column pulse electrochemical detection of amino acids

A novel electrophoresis technique, in which the separation column was replaced by a strip of Nafion membrane (5.0 cm x 0.20 mm x 0.25 mm), was developed for the separation of an amino acid mixture (glycine, asparic acid and lysine), followed by quadruple-pulse electrochemical detection. Nafion membrane contains hydrophilic pores (10-20 A and 50-60 A in size) acting as very narrow electrophoresis channels. The fixed-charge sites (-SO(3) (-)) on the hydrophilic pore surface provide a strong charged background. A platinum disk electrode (0.90 mm inner diameter) was employed as the detection electrode and the electrophoresis cathode was used as the quasi-reference and counter electrode for the end-column electrochemical detector, without decoupler. Under optimized conditions the mixture of amino acids could be separated at a voltage of only 90 V with a detection limit of 10(-7) M, indicating that Nafion membrane electrophoresis is a potentially attractive technique for the separation of small organic molecules or ions.     

An interface chip connection between capillary electrophoresis and thermal lens microscope

A thermal lens microscope (TLM) detection of capillary electrophoresis (CE) utilizing microchip technology was developed. Fused-silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel in a microchip. The detection limit by TLM was estimated as 2.8 x 10(-7) absorbance by measuring pure water. The detection limit of derivatized amino acids determined by CE-TLM was estimated as 2.4 x 10(-8) M, which was 100 times lower than that of conventional absorbance detection.     

Sub-femtomole determination of DABSYL-amino acids with capillary zone electrophoresis separation and laser-induced thermo-optical absorbance detection

Fifteen amino acids are determined as the DABSYL (4-4-dimethylaminoazobenzene-4'-sulfonyl chloride) derivative with capillary zone electrophoresis separation and low-power laser-induced thermo-optical detection. Separation efficiencies are on the order of 200 000 theoretical plates and the detection limit, 3 s, is 200 attomole of glycine injected onto the column. At the detection limit, 0.7 attomole of glycine is present within the 40 picoliter detection volume.     

Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette

Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.     

Rapid detection of proteins by enzyme-linked immunofiltration assay after transfer onto nitrocellulose membranes.

Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.     

Die Primereinschluß-Technik zum Enzymnachweis bis 10−12g nach Polyacrylamid-Elektrophorese,dargestellt an Phosphorylasen

Inclusion of primers into polyacrylamide makes possible a highly sensitive detection (down to 10^?12 g) of enzymes, after gel electrophoresis, if the gel is incubated with activated subunits of the macromolecule to be synthesized. For phosphorylases and glycogen as primer, incubation with glucose-1-phosphate will yield starch where the enzymes are located. For polynucleotidephosphorylases and s-RNA as primer, nucleic acids are synthesized from adenosinediphosphate in the presence of Mg²⁺.     

Chromatographic amino acid analysis of protein hydrolysed in polyacrylamide after removal of ammonia

Summary A simple method for the removal of NH₃ from amino acids is presented. The method is based on a cation-exchange resin from which amino acids are eluted with NH₄OH. The eluent is then removed under reduced pressure. The method allows the ninhydrin-based detection of amino acids after hydrolysis of stained protein bands in polyacrylamide gels. This was previously not possible since NH₃ produced by the hydrolysis of polyacrylamide interferes with the ninhydrin-detection of basic amino acids. The method should also be applicable to the detection of amino acids with o-phthalaldehyde.     

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.     

Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection

A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.     

CMC-g-DNA接枝共聚物的合成与表征

在磷酸盐缓冲溶液中用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)活化羧甲基纤维素钠(CMC)侧链上的羧基; 在室温下再将活化的CMC与5'端经氨基修饰的单链脱氧核糖核酸(DNA)齐聚物(ODNs)反应, 获得CMC上接枝ODNs的共聚物(CMC-g-ODNs), 以Lambda DNA为模板, 通过聚合酶链式反应(PCR), 将接枝的ODNs扩增为长度为1300个碱基对的双链DNA, 从而制得CMC侧链上接枝DNA的共聚物CMC-g-DNA. 采用傅里叶红外光谱仪测定CMC与NHS形成的中间体; 用水平式琼脂糖凝胶电泳和垂直板变性聚丙烯酰胺凝胶电泳对CMC-g-DNA接枝共聚物进行表征. 结果表明, 合成了CMC-g-DNA接枝共聚物, 且在酸性条件下CMC的活化效果更好; 同时, 接枝在CMC上的DNA在琼脂糖凝胶电泳中迁移速率加快, 而在聚丙烯酰胺凝胶电泳中迁移速率减慢.     

Simultaneous concentration enrichment and electrophoretic separation of weak acids on a microchip, using in situ photopolymerized carboxylate-type polyacrylamide gels as the permselective preconcentrator

A method for the simultaneous concentration and separation of weak acids using an acidic polyacrylamide gel, fabricated in the microfluidic channel of a commercial poly(methyl methacrylate)-made microchip, is reported. This approach is based on simple photochemical copolymerization for the fabrication of a permselective preconcentrator. The intersection of the poly(methyl methacrylate)-made microchip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide, and 2-acrylamidoglycolic acid, with riboflavin as a photocatalytic initiator. In situ polymerization, near the cross of the sample outlet channel, was performed by irradiation with an argon ion laser beam that is also used as the light source for fluorimetric detection. The electrokinetic properties, combined with electrostatic repulsion between sample components and the anionic groups on the polyacrylamide gel, enable the entrapment and concentration of weak acids at the interface of the cathodic side of the gel plug. This method displays concentration factors of up to 10(5) within 3 min. The effectiveness of the ionic preconcentrator was demonstrated by the sensitive analysis of fluorescein isothiocyanate-labeled amino acids.     

Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation-element mass spectrometry with phosphorus-31 detection.

Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with (31)P detection has been used for spotting of phosphoproteins after one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and membrane transfer. By analyzing a mixture of myoglobin, alpha-casein and reduced fibrinogen it is demonstrated that phosphoproteins are specifically recognized by this method. A special washing step was found to be necessary to remove phosphate noncovalently bound to proteins. The (31)P signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the (31)P signal from a single laser ablation trace by the total amount of phosphoprotein applied to the gel, a detection limit of 5 pmol of phosphorus is estimated.     

Degradation of individual intracellular proteins analyzed by two-dimensional gel electrophoresis and computerized video densitometry

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse-chase-labeling of cells using radioactive amino acids [35S]methionine, two-dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two-dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell-type-specific proteins were significantly enhanced (p less than 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.