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1.
A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.  相似文献   

2.
Immunoblotting is a commonly used technique for the immunodetection of specific proteins which have been fractionated by polyacrylamide gel electrophoresis. We describe here a simple procedure for the double staining of immunoblots, first to detect the immunoreactive component(s) by histochemistry using enzyme-conjugated secondary antibodies, and second to visualize the general protein electrophoretogram using India ink. This procedure permits the direct comparison of electrophoretic mobilities between the immunoreactive protein(s) and the total protein population as well as protein standards of known Mr. The experimental advantage of the procedure is that no additional manipulation of the protein samples or the standards is necessary prior to electrophoretic fractionation. In this report, detection of the vitamin D-dependent calcium-binding protein, calbindin-D28K, is used to illustrate the application of the procedure.  相似文献   

3.
A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.  相似文献   

4.
A method is described for determination of protein in biological preparations containing various interfering substances normally present in extraction media. The main steps of the procedure consist in depositing the protein solution on filter paper strips and removing all small molecular weight substances by washing with a number of aqueous and nonaqueous solvents. The protein remaining on the paper is then determined by a modification of Lowry's colorimetric procedure. The method also permits the determination of protein in radioactive samples which have been previously counted using a liquid scintillation mixture such as Apuasol or toluene-POPOP.  相似文献   

5.
Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.  相似文献   

6.
Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.  相似文献   

7.
Computation of the dipole moments of proteins.   总被引:1,自引:0,他引:1       下载免费PDF全文
A simple and computationally feasible procedure for the calculation of net charges and dipole moments of proteins at arbitrary pH and salt conditions is described. The method is intended to provide data that may be compared to the results of transient electric dichroism experiments on protein solutions. The procedure consists of three major steps: (i) calculation of self energies and interaction energies for ionizable groups in the protein by using the finite-difference Poisson-Boltzmann method, (ii) determination of the position of the center of diffusion (to which the calculated dipole moment refers) and the extinction coefficient tensor for the protein, and (iii) generation of the equilibrium distribution of protonation states of the protein by a Monte Carlo procedure, from which mean and root-mean-square dipole moments and optical anisotropies are calculated. The procedure is applied to 12 proteins. It is shown that it gives hydrodynamic and electrical parameters for proteins in good agreement with experimental data.  相似文献   

8.
Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure.  相似文献   

9.
A short procedure for the isolation of band-3 protein, the protein responsible for anion exchange in erythrocytes, in a reasonable degree of purity was developed. Using this protein preparation and a novel procedure for membrane-protein reconstitution, vesicles displaying the basic features of the anion-exchange system of the erythrocyte were obtained. The reconstitution procedure is based on slow direct removal of Triton X-100 from aqueous lipid/detergent solutions. According to the composition of the reconstitution medium, either small single-walled or large multi-walled vesicles are obtained. The procedure conserves protein properties well, as is revealed by the similarity of the rates of SO4(2-) exchange in erythrocytes and reconstituted vesicles when corrected for the relevant volumes. A number of functional features of the exchange system were studied and compared with those of the native membrane.  相似文献   

10.
A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.  相似文献   

11.
本文介绍一种碱性磷酸酶标A蛋白加强的PAP技术。采用PAP技术、碱性磷酸酶标A蛋白(PAAP)技术PAAP和加强的PAP(PAP-PAAP)技术显示下丘脑室旁核催产素(OT)能神经元。结果发现,其中使用PAP-PAAP技术免疫反应产物的显色最深。此技术的原理可能是,由于A蛋白分子至少有四个位点能与IgG分子的Fc段高亲合性地结合,故在该技术中,先经过PAP程序的三步免疫反应并显色后,每个与一抗结合的二抗分子上和每个与二抗结合的PAP复合物分子上各暴露一个能与A蛋白分子结合的Fc段,在随后经过PAAP技术处理时,部分PAAP复合物分子就结合在这些Fc段上,经显色后,PAAP技术显示的浅紫兰色与PAP技术显示的浅棕褐色重叠,变成更深的反差明显的深棕褐色。  相似文献   

12.
We recently reported a highly sensitive assay for measuring protein in solution based on the capacity of glutaraldehyde-treated protein to bind silver. This assay has now been made more sensitive, with a lower limit of detection of 5 ng, and more reproducible by supplementing protein samples with sodium dodecyl sulfate (SDS) to reduce protein loss to glassware. Two procedures have been developed. In one, protein samples are supplemented with both SDS and Tween 20 to yield very steep protein dose-response curves, which allow for more precise protein determinations, and very stable color formation, permitting OD measurements to be made several hours after the assay has been completed. In the second procedure, protein samples are supplemented with SDS alone which results in a less steep dose-response curve and less stable color formation but makes the assay substantially more tolerant of interfering substances. Thus, proteins in most commonly used buffers can be assayed directly with the second procedure without the need for buffer exchange. The procedure of choice, therefore, depends on the type and concentration of interfering substance. Proteins in buffers totally incompatible with either assay procedure (e.g., those containing reducing agents) can be easily buffer exchanged by centrifugation through 0.2% SDS equilibrated, drained Bio-Gel P-2 beads. The clinical utility of this improved assay is demonstrated by the accurate quantitation of protein in 0.5 μl of samples of human cerebral spinal fluid. This assay should therefore prove especially useful when a limited amount of protein is available for quantitation.  相似文献   

13.
Abstract

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.  相似文献   

14.
Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.  相似文献   

15.
Protein 4.1 is a multifunctional structural protein occupying a strategic position in the erythrocyte membrane. It is present in the erythrocyte membrane skeleton and in many nonerythroid cells. This report describes a novel method for purifying this protein based on its selective interaction with inositol hexaphosphate dimagnesium tetrapotassium salt. This interaction was discovered in the course of chromatography of high-salt extract of inside-out membrane vesicles on Procion orange MX-2R-Sepharose. The new procedure is simple and selective and produces protein 4.1 with better yield than that obtained with a previously published procedure. The purified protein 4.1 has the same immunoreactivity and the same alpha-chymotryptic digest profile as protein 4.1 purified by published methods and is fully functional in enhancing the interaction between F-actin and spectrin dimers.  相似文献   

16.
A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.  相似文献   

17.
A batchwise purification procedure of neurofilament proteins   总被引:6,自引:0,他引:6  
A rapid batchwise purification procedure for neurofilament proteins from bovine spinal cord is described. A crude filament fraction can be obtained by treating the tissue with Triton X-100, followed by centrifugation through sucrose. From this crude filament fraction the protein is processed through a batch purification procedure using hydroxyapatite in 8 M urea. With this procedure, approximately 0.5 g of purified neurofilament protein is obtained from a single bovine spinal cord in less than 3 days.  相似文献   

18.
目的:通过优化现有的蛋白芯片检测过程,在保证检测准确性的同时缩短甲胎蛋白(Alpha Fetal protein,AFP)的检测时间,提高检测效率,为原发性肝癌的筛查提供经济、便捷、省时、有效的检测方法。方法:本研究在传统蛋白芯片检测流程(1-1.5小时)的基础上,通过优化检测流程将检测时间缩短至18分钟,并且通过和传统方法进行比较,评价该优化方法的检测效能。结果:与传统蛋白芯片检测方法相比,本优化方法的检测时间缩短至18分钟。重复检测同一样本,传统方法 AFP水平为16.50±1.172ng/m L,优化方法 AFP水平为18.33±1.029 ng/m L,结果无明显统计学差异(P=0.251)。结论:本研究成功地优化了AFP的蛋白芯片检测流程,在缩短检测时间的同时,保证了检测的准确率,是一种经济省时易操作的AFP检测方法。  相似文献   

19.
The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.  相似文献   

20.
蛋白提取液经加热后,流经一个DEAR-纤维素小柱,然后通过透析或稀释均可得到结晶。如在匀浆时加入足够的711阴离子交换树脂,则蛋白提取液加热后可直接透析结晶,连DEAR-纤维素小柱也可以不用。席法省略了耗费时间的超滤浓缩和粗大的凝胶柱装置。  相似文献   

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