一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶原为材料,探索一种从SDS-PAGE胶上回收蛋白质做MALDI-TOF质谱的方法.所用的回收方法包括电洗脱、脱盐、除SDS等过程.电洗脱用的是高盐阻断法,脱盐用的是超滤技术,去除SDS用的是冷丙酮沉淀法.结果证明,此方法至少对一些蛋白质（如尿激酶原和牛血清白蛋白）是可行的.     

苏云金杆菌4.0718菌株杀虫晶体65kD原毒素的质谱分析

以苏云金杆菌4.0718菌株杀虫晶体65kD原毒素为材料,探索了从SDS－PAGE胶上回收蛋白质进行质谱分析的方法。蛋白纯化的步骤包括SDS－PAGE、电洗脱、脱盐、除SDS。电洗脱采用半透膜法,用超滤法脱盐,冷丙酮沉淀法除去SDS。结果表明,纯化的65kD原毒素经ESI－MS检测,分子量约64500Da。经MALDI－MS检测,未能有明显蛋白峰出现,这可能是该蛋白由于较强的疏水作用,溶解性差,在与基质混合时处于聚和悬浮态所致。     

SDS—聚丙烯酰胺凝胶电泳纤维蛋白自显影法检测不同分子…

ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）纤维蛋白显影方法是经ＳＤＳ－ＰＡＧＥ将不同分子量的纤溶酶原激活剂（ＰＡ）分开,然后再转溶纤维蛋白板使之产生溶带而检测ＰＡ物质活性及分大小的方法,此法与Ｗｅｓｔｅｒｎ印迹方法此具有检测更简便、灵敏、快速的特点,且是活性检测,但其分子量分析精度不如Ｗｗｅｓｔｅｒｎ印迹法。本研究用ＳＤＳ－ＰＡＧＥ纤维蛋白自显影方法对本室表达的生组组织型－ＰＡ及突体Ｎ１１７Ｑ     

SDS－聚丙烯酰胺凝胶电泳纤维蛋白自显影法检测不同分子量的纤溶酶原激活剂

ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）纤维蛋白显影方法是经ＳＤＳ－ＰＡＧＥ将不同分子量的纤溶酶原激活剂（ＰＡ）分开,然后再转溶纤维蛋白板使之产生溶带而检测ＰＡ物质活性及分子大小的方法。此法与Ｗｅｓｔｅｒｎ印迹方法比具有检测更简便、灵敏、快速的特点,且是活性检测,但其分子量分析精度不如Ｗｅｓｔｅｒｎ印迹法。本研究用ＳＤＳ－ＰＡＧＥ纤维蛋白自显影方法对本室表达的重组组织型－ｐＡ（ｒｔ－ＰＡ）及突变体Ｎｌｌ７Ｑ／Ｎｌ８４Ｑ／△（Ｋ２９６——Ｇ３０２）及标准尿激酶（ＵＫ）和ｒｔ－ＰＡ进行了分析鉴定。     

酰化人红细胞超氧化物歧化酶稳定性的研究

用月桂酸对人红细胞超氧化物歧化酶（ｈ－ＳＯＤ）进行化学修饰得到酰化ｈ－ＳＯＤ（Ａｃ－ｈＳＯＤ）,并对Ａｃ－ｈＳＯＤ和ｈ－ＳＯＤ的稳定性进行了比较。结果表明：Ａｃ－ｈＳＯＤ活力为ｈ－ＳＯＤ的７２％。比活力为４０００Ｕ／ｍｇ,Ａｃ－ｈＳＯＤ的热稳定性、酸碱稳定性及抗蛋白酶水解能力均比天然酶提高。     

地衣芽孢杆菌β—甘露聚糖酶的纯化及其性质的研究

地衣芽孢杆菌１Ｂａｃｉｕｕｓ Ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＢＬ－３０６产生的胞外β－甘露聚糖酶经硫酸铵分级盐析,ＤＥＡＥ－纤维素柱层析。Ｓｅｐｈａｄｅｘ－Ｇ１００柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化,得到ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）均一样品。用ＳＤＳ－ＰＡＧＥ测得纯化后β－甘露聚糖酶分子量为２６０００道尔顿。用凝胶等电聚焦电泳（ＰＡＧＥＩＥＦ）测得等电点ＰＩ为５．０。该酶     

酰化人红细胞超氧化物歧化酶稳定性的研究

用月桂酸对人红细胞超氧化物歧化酶进行化学修饰处到酰化ｈ－ＳＯＤ,并对Ａｃ－ｈＳＯＤ和ｈ－ＳＯＤ的稳定性进行了比较。结果表明：Ａｃ－ｈＳＯＤ活力为ｈ－ＳＯＤ的７２％,比活力为４０００Ｕ／ｍｇ,Ａｃ－ｈＳＯＤ的热稳定性,酸碱稳定性及抗蛋白酶水解能力均比天然酶提高。     

植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术

本研究建立和改进了检测番木瓜和西葫芦组织粗汁液里的番木瓜环斑病毒（ＰＲＶ）的ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法。用不同的ＥＬＩＳＡ方法来检测不同寄主植物粗汁液里的ＰＲＶ,其所用的合适的制备粗汁液的缓冲液是不同的。用ＤＡＣ－ＥＬＩＳＡ法检测西葫芦粗汁液时,以０．５ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ７．５,内含０．１ｍｏｌ／Ｌ乙二胺四乙酸二钠）为宜;而检测番木瓜粗汁液时,则还要加入０．２５ｍｏｌ／Ｌ脲。用Ｄｏｔ－ＥＬＩＳＡ法检测时,在上述磷酸盐缓冲液中加入２％聚乙烯吡咯烷铜能提高对西葫芦粗汁液的检测效果。应用合适的制备粗汁液的缓冲液,ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法的灵敏度分别提高到１／４０９６和１／１０２４（稀释度）。本研究还表明,影响ＤＡＣ－ＥＬＩＳＡ法的定过测定的主要因素是粗汁液的稀释度和包被液（０．０５ｍｏｌ／Ｌ碳酸盐缓冲液,ｐＨ９．６）的用过。在较高粗汁液稀释度和包被液的用量相同时,粗汁液里的病毒含量与ＤＡＣ－ＥＬＩＳＡ法的ＯＤ４９２ｎｍ值呈真实的线性关系。     

多胺对水稻CMS系及其保持系幼穗蛋白质,核酸和活性氧代谢的影响

用Ｄ－Ａｒｇ＋ＭＧＢＧ处理保持系,降低花粉可育度,并使其幼穗中蛋白质、ＤＮＡ和ＲＮＡ含量以及蛋白酶、ＲＮＡ酶和ＤＮＡ酶活性下降,使Ｏ２生成速率和ＭＤＡ含量上升。Ｐｕｔ＋Ｓｐｄ＋Ｓｐｍ有除或部分消除Ｄ－Ａｒｇ＋ＭＧＢＧ的上述效应（对酶活性的除外）。Ｄ－Ａｒｇ＋ＭＧＢＧ也使ＰＯＤ、ＳＯＤ和ＣＡＴ活性上升,但是,用Ｐｏｔ＋Ｓｐｄ＋Ｓｐｍ处理不育系,使花粉可育度轻度提高,并使其幼穗蛋白质、ＤＮＡ和ＲＮＡ含     

家蚕滞育生物钟蛋白质Ease A4的纯化及其分子结构分析

ＥａｓｅＡ４是家蚕卵的一种滞育生物钟蛋白质．产下后２ｄ的家蚕Ｃ１０８品种滞育性卵,经过丙酮脱脂、８５℃热处理、硫酸铵沉淀和ＳｅｐｈａｄｅｘＧ－２５凝胶过滤层析初步分离,进一步经过Ｓｅｐ－ＰａｋＣ１８脱盐浓缩,ＨＰＬＣ（柱为ＹＭＣ－ＰａｃｋＰｒｏｔｅｉｎ－ＲＰ）分离,通过ＳＤＳ－ＰＡＧＥ和ＭＡＬＤＩＭＳ方法鉴定,纯化得到ＥａｓｅＡ４蛋白质．从１０ｇ蚕卵最终得到了１１．８μｇＥａｓｅＡ４．ＥａｓｅＡ４由从Ｈｉｓ到Ｔｙｒ的１５５个氨基酸残基构成,蛋白质部分的分子量为１６６０１．其２２位氨基酸残基Ａｓｎ处有一个Ａｓｎ－Ｘ－Ｔｈｒ糖基化场所,并有糖基结合在该部位,糖基的分子量约为７６０．ＥａｓｅＡ４的６１位和１５０位的两个Ｃｙｓ氨基酸残基之间存在二硫键．糖基和二硫键的存在不仅有利于酶蛋白的分离,还可能与酶活性有关     

Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate-polyacrylamide gels and polyvinyldifluoride membranes

The resolving power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with isoelectric focusing in two-dimensional gel electrophoresis has made it one of the most important techniques for resolving complex mixtures, and it is of great importance for proteome mapping projects. As a result of this, methods for postelectrophoretic protein characterization are of great interest as exemplified by in situ protease digestion combined with mass spectrometry (MS), which is the method of choice for identification of proteins. In this study we have developed and compared methods for recovering intact proteins from polyacrylamide gels and electroblotting membranes to define efficient methods compatible with MS. These methods complement in situ digestion protocols and allow determination of the molecular mass of whole proteins separated by SDS-PAGE. Passive elution of proteins from SDS-PAGE gels was efficient only in the presence of SDS, whereas electroelution was achieved using buffers without SDS. Surface-enhanced laser desorption/ionization MS (SELDI-MS) analysis of proteins eluted in the presence of SDS was possible using ion exchange ProteinChip arrays for concentration of sample and removal of SDS. Comparison of different electroblotting methods verified that the different membranes and buffers were equally efficient for transfer of proteins in the range 20-100 kDa. Elution from polyvinyldifluoride membranes was most efficient using either concentrated solutions of trifluoroacetic acid (TFA) or combinations of 8M urea and 1% Triton X-100, 1% Tween 20, or 40% isopropanol. The same result was obtained using nitrocellulose membranes, except that these were incompatible with organic solvent and TFA. Elution by TFA was compatible with matrix-assisted laser desorption/ionization MS (MALDI-MS) but was complicated by a high degree of trifluoroacetylation of the proteins. Alternatively, elution by 8M urea+1% Triton X-100, 1% Tween 20, or 40% isopropanol was compatible with both SELDI-MS and MALDI-MS. Eluted proteins were identified in MS experiments by intact mass determination, by peptide mapping, and by MS/MS analysis.     

从琼脂糖电泳凝胶中回收DNA的几种简便方法

介绍两类从普通琼脂糖电泳凝胶中回收ＤＮＡ的简便、快捷、高效且廉价的方法．第一类为电泳洗脱法．方法ａ：利用１．５ｍＬ微量离心管、ｌｍＬ吸头、尼龙网膜和透析膜做成的一个小装置,快速有效回ＤＮＡ,最终回收率为７０％左右．方法ｂ：不用ＤＥＡＥ－纤维素膜,而用透析膜在凝胶中作出横隔挡在ＤＮＡ条带前,最终回收率为５０％左右;第二类为冰冻融解法,最终回收率也在５０％左右．如果联合使用冰冻融解法和电泳洗脱法,回收率可进一步提高至９０％．     

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.     

蜘蛛HMW-gDNA的电洗脱纯化提取

该文研究了蜘蛛大分子量基因组DNA（HMW-gDNA）的提取以及一种高效电洗脱纯化装置的构建。以蜘蛛胸部肌肉组织为原料,通过自改良CTAB法提取蜘蛛HMW-gDNA,利用透析膜和2 mL离心管构建一种新的HMW-gDNA快速凝胶回收装置,并对蜘蛛HMW-gDNA进行电洗脱分离回收。结果显示,改良CTAB法可高效提取蜘蛛HMW-gDNA（>48.5 kb）,且通过透析膜的截留作用,对普通琼脂糖凝胶中目的HMW-gDNA进行快速电洗脱分离,其回收率超过75%,OD₂₆₀/OD₂₈₀处于1.8～2.0之间,对HMW-gDNA完整性无影响。综合结果表明, 改良CTAB法可用于蜘蛛HMW-gDNA的提取,此电洗脱纯化装置可从普通琼脂糖中高效回收HMW-gDNA,是一种低成本、简捷、高效且实用性强的凝胶回收方法。     

Affinity chromatographic procedure for the quantitative recovery of DNA fragments from agarose gels

A simple and reliable method for the recovery of specific fragments of DNA from agarose gels is presented. The electroelution of the DNA onto the NENSORB cartridge matrix with the subsequent elution of the bound DNA by a methanol (50% v/v) wash has been shown to result in the quantitative recovery of the restriction fragment. Of importance is the fact that the DNA purified by this procedure is a viable substrate for further digestion by a second restriction endonuclease. The method does not require either phenol extraction or extensive desalting of the sample.     

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.     

Removal of sodium dodecyl sulfate from proteins.

A convenient and relatively simple electrodialysis method for the removal of sodium dodecyl sulfate (SDS) from proteins is described. Six samples can be processed simultaneously. The kinetics of removal of SDS from proteins by equilibrium dialysis and electrodialysis have been studied.