螺旋藻多糖的琼脂糖凝胶电泳鉴别

目的用琼脂糖凝胶电泳对螺旋藻多糖进行定性鉴别。方法利用螺旋藻多糖与肝素、硫酸软骨素、标准硫酸化的葡聚糖之间电荷密度及分子量的差异 ,在巴比妥缓冲液中进行琼脂糖凝胶电泳。结果根据电泳迁移率 ,可明显区分螺旋藻多糖与其他多糖。结论该法重现性较好 ,操作简单 ,可用于螺旋藻多糖的定性鉴别     

等电点除蛋白的pH值对螺旋藻酸性多糖硫酸基团含量的影响

考察等电点除蛋白对螺旋藻酸性多糖的硫酸根含量的影响。采用热碱水提取，乙醇沉淀，CPC络合得螺旋藻酸性多糖。硫酸苯酚法测定糖含量，聚丙烯酰胺凝胶电泳和氯化钡明胶法检查硫酸根，200～400nm紫外扫描及水解氨基酸检测蛋白。结果表明，不同酸化条件对螺旋藻酸性多糖的硫酸根含量有明显的影响，以pH值4．0去除蛋白效果最佳，硫酸根保留相对较多。     

一步等电聚焦纯化羊血铜锌超氧化物歧化酶

本文用水平板制备等电聚焦电泳纯化了羊血红细胞铜、锌超氧化物歧化酶。其粗酶液经过一步等电聚焦比活力由271.6u/mg蛋白提高到6254u/mg蛋白,纯化倍数提高了22.2倍,活力回收72.9％。聚丙烯酰胺凝胶电泳及薄层聚丙烯酰胺等电聚焦电泳均呈一条带,等电点为pH6.8。     

螺旋藻中性多糖硫酸化及性质研究

采用氯磺酸-吡啶法对螺旋藻中性多糖进行硫酸化修饰；玫瑰酸钠法测定修饰前后的多糖SO4^2-含量分别为0．17％、20．0％；取代度Ds=0．429；利用琼脂糖凝胶电泳对多糖硫酸酯修饰物进行研究。结果表明多糖硫酸酯修饰物的琼脂糖凝胶电泳呈一均一斑点．与其他几种硫酸化多糖相比迁移率有较大差异；红外光谱法确定硫酸酯键在1270～1200cm^-1．1000～800cm^-1处存在强的特征吸收峰；HPLC显示出一个主要的单峰，说明此制备方法可行。     

重组人碱性成纤维细胞生长因子等电聚焦分析

目的：建立重组人成纤维细胞生长因子的等电聚焦电泳法。方法：利用载体两性电解质和聚丙烯酰胺,采用毛细管灌胶不自制平板胶,并从凝胶浓度、电极溶液等方面优化重组人成纤维细胞生长因子的等电聚焦电泳条件。结果：pH梯度曲线线性因归方程为Y＝－1．2593X＋10．808,γ=0.9939。重组人成纤维细胞生长因子的等电点为9．4（n=5,RSD=1.0％）。结论：该法简便,实用,重复性好,为碱性多肽或蛋白的等电点测定提供了实验依据。     

硫酸多糖电泳方法的研究

目的研究几种海洋硫酸多糖的电泳行为。方法采用醋酸纤维膜电泳法和聚丙烯酰胺凝胶电泳(PAGE)法 ,染色剂选用爱茜蓝。结果几种海洋硫酸多糖的醋酸纤维膜电泳均呈单一斑点 ,与标准肝素相比 ,几种海洋硫酸多糖的迁移值不同 ;在建立的PAGE电泳条件下肝素获得了很好的分离 ,几种海洋硫酸多糖的PAGE电泳区带均呈连续分布 ,并利用电泳图象扫描软件进行了相对分子量分布分析。结论醋酸纤维膜电泳法可用于海洋硫酸多糖的定性鉴别 ,PAGE法可用于海洋硫酸多糖相对分子量分布的测定     

β—环糊精对SOD的化学修饰

牛血铜锌－超氧化物歧化酶（Ｃｕ，Ｚｎ－ＳＯＤ）用β－环糊精修饰后，其理化性质发生了部分变化：修饰后的酶呈不均一性，在聚丙烯酰胺凝胶电泳出现４条带，在薄层平板等电聚焦电泳中，等电点降低，修饰酶的紫外吸收光谱未见明显改变，抗蛋白酶水解能力增加。     

应用萃取-超滤两步法从过期人血中提纯人血红蛋白

目的：建立一种有效的实验方法，从过期的全血及血液制品（少浆全血；浓缩红细胞；洗涤红细胞）中，提取人天然无基膜血红蛋白（SHFb）。方法：采用甲苯萃取法有效去除血液中的有形成分（细胞膜等），再采用Millipore超滤装置分离提纯人血红蛋白，得到SFHb，然后建立合理的实验方法（电泳、紫外－可见光扫描、反相高效液相，库尔特血液分析系统和ABO血型凝集试验等），确定纯化产品的产率、浓度、纯度。结果：应用上述方法，测得SFHb的浓度为15．2g/dl；纯度为98．3％；血型 凝集实验：阴性；紫外扫描图谱、反相高效液相峰谱与对照品吻合；聚丙烯酰胺凝胶电泳结果显示，产品呈均一分布，90％电泳迁移率近似Marker(45KD);15%SDS-聚丙烯酰胺凝胶电泳结果显示确定了血红蛋白蛋白质的亚基组成及分子量,电泳迁移率与对照品完全一致。     

蝮蛇毒纤溶酶的分离纯化及其制剂的临床应用

目的:采用凝胶电泳法分离纯化长白山白眉蝮蛇毒纤溶酶并将其制剂应用于临床。方法:采用DEAE- Sepharose CL-6B和Heparin CL-6B层析方法,从蝮蛇毒中分离纯化纤溶酶,通过临床应用分析成品制剂的治疗效果。结果:蝮蛇毒纤溶酶经HPLC为单一峰,等电聚焦电泳为一条带,其等电点为4．55,经SDS-聚丙烯酰胺凝胶电泳测得分子量为29．4kD,将其成品制剂应用于临床,结果表明有效减少了缺血性脑血管病血栓的形成,使缺血部位迅速恢复功能。     

辽宁产东亚钳蝎毒中哺乳动物毒素Ⅲ的分离纯化及部分性质的研究

采用 CM—Sephadex C—50,SP—Sephadex C—25离子交换柱层析和 SephadexG—50凝胶过滤三步分离程序,从东亚钳蝎毒中得到一种新的哺乳动物毒素。经低 pH 系统不连续聚丙烯酰胺凝胶园盘电泳,SDS—不连续聚丙烯酰胺凝胶板电泳及等电聚焦聚丙烯酰胺凝胶园盘电泳鉴定说明该毒素为电泳纯的蛋白质。用 SDS 电泳法测得其分子量为8,750道尔顿,等电聚焦电泳法测定 pH 为8.2。纯化成份的产率为2.5%。     

Gradient polyacrylamide gel electrophoresis for determination of molecular weights of heparin preparations and low-molecular-weight heparin derivatives.

The M(r) values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of M(r) determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the M(r) values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average M(r), weight-average M(r), and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the M(r) values of heparin and LMW heparin.     

Analysis and determination of biological activity of short-chain peptides from porcine brain hydrolysate

Gel permeation chromatography fractions of short-chain peptides from a hydrolysate product, which in turn was from the purified porcine brain through enzyme hydrolysis, were tested for their biological activities. The results showed that the fractions A4 and A5 had significant biological activities. The two fractions were analyzed with analytical techniques such as high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preliminary results showed that the main components of these two fractions were short-chain acidic peptides with a relative molecular mass (M(r)) of less than 2400.     

Improved detection methods significantly increase the detection window for EPO microdoses

To reproduce a potential doping scenario, a 2 week administration of recombinant erythropoietin (rEPO) microdoses alone or in combination with growth hormone (GH) microdoses (three times a week) was performed on healthy and athletic male subjects. The aim of this study was to evaluate the identification capability of rEPO in samples obtained during and post treatment. Detection was tested in urine and blood using the antidoping techniques for rEPO detection (iso-electric focusing (IEF)-, sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and for some urine samples the sarcosyl (SAR)-PAGE method) with some improvements: for blood samples, instead of a simple concentration step, immuno-extraction of EPO was performed for all urines to limit protein contamination that can affect migration. In addition, elution buffer modifications also improved the quality of migration. The use of a recently validated biotinylated anti-EPO antibody simplified the protocols, allowing a single transfer step instead of a double-blot even by IEF with a lowered background. The criteria for suspicious blood and urine samples by IEF were also re-evaluated. While endogenous EPO was not decreased over the course of the study, EPO microdoses were detectable in blood and urine between 24 h and 72 h after an administration. Detection in urine in combination with SDS-PAGE was the most sensitive combination for prolonged detection (100% identification after 48 h, 91% after 72 h), slightly better than IEF. Urine samples also tested by SAR-PAGE indicated a similar sensitivity of detection to SDS-PAGE. GH co-administration had no impact on rEPO elimination/detection.     

一个Rett综合征家系MECP2基因突变分析及X染色体失活研究

目的:研究Rett综合征(RTT)患者的MECP2基因突变,探讨X染色体失活在RTT致病机制中的作用.寻找临床基因诊断Rett综合征的实验方法.方法:首先采集Rett综合征先证者及家系成员的外周血提取基因组DNA,聚合酶链式反应扩增RTT致病基因MECP2的3个外显子和3′UTR(3′端非翻译区),琼脂糖凝胶电泳和非变性聚丙烯酰胺凝胶电泳分离目的片段,PCR产物经纯化后直接测序.应用甲基化PCR(methylation specific PCR,MSP)进行X染色体失活分析.结果:发现患儿MECP2基因的Exon3发生1个点突变C473T(T158M),该突变为新生突变.患儿与患儿母亲表现为非随机X染色体失活.结论: X染色体非随机失活在RTT的致病机制中发挥某种作用,C473T(T158M)点突变可作为典型Rett患者基因诊断的位点之一.     

Quantitation of myosin light chain phosphorylation in intact smooth muscle.

An improved method for quantitating the extent of myosin light chain (P-LC) phosphorylation in small smooth muscle samples is described. Native myosin was isolated from other cellular proteins in a crude supernatant fraction prepared from a few milligrams of bovine tracheal smooth muscle by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium pyrophosphate (PPi). When potassium iodide (KI, 0.6 M) was added to the crude supernatant fraction, myosin migrated into the gels during electrophoresis. Without adding KI, myosin remained at the top of the gel so that the myosin content in the gel was 20 times less than in the presence of KI. After the PPi-PAGE, myosin was subjected to isoelectric focusing (IEF) on polyacrylamide slab gels to separate the phosphorylated from the nonphosphorylated forms of the P-LC. The extent of P-LC phosphorylation was quantitated after densitometric scanning of silver-stained IEF gels. Examination of the temporal changes in carbachol-induced contraction and P-LC phosphorylation in tracheal smooth muscle strips exhibited a relatively transient change in the P-LC phosphorylation as shown in other smooth muscle preparations. This procedure is applicable to investigations on the role of Ca2+.calmodulin-induced activation of myosin light chain kinase and phosphorylation of smooth muscle myosin.     

A pilot study comparing the purity, functionality and isoform composition of alpha-1-proteinase inhibitor (human) products

OBJECTIVE: Alpha-1-proteinase deficiency predisposes affected individuals to early onset pulmonary emphysema, and is treated with an alpha-1-proteinase inhibitor (A1-PI) from pooled human plasma. The objective of this pilot study was to assess analytical parameters of the three A1-PI products (Aralast, Prolastin, Zemaira) that may impact on clinical efficacy, safety, and convenience. These included: purity of the preparation; nature of impurities; functionality; and isoform composition. METHODS: Purity was evaluated using reverse phase and size exclusion chromatography high performance liquid chromatography (RP-HPLC and SEC-HPLC), capillary zone electrophoresis (CZE), sodium dodecyl sulfate polyacrylamide gel electrophoresis, sodium dodecyl sulfate capillary gel electrophoresis and Western blot analysis. The identity of protein impurities was determined by immunonephelometry; functionality by calculating the ratio of mg active A1-P1 present (by anti-neutrophil elastase activity assay) to the mg antigenic A1-PI (by immunonephelometry); and normality of the A1-PI isoform pattern by isoelectric focusing (IEF). Three samples of Zemaira and one sample each of Aralast and Prolastin were available for analysis. RESULTS: Zemaira had the highest specific activity. Using RP-HPLC analysis Zemaira averaged 99% purity, Aralast 70% and Prolastin less than 62%. Using SEC-HPLC Zemaira was 95.98% monomeric, Prolastin 79.00% and Aralast 63.55%. Prolastin had lower activity/mg antigenic A1-PI than the other two products. A shift in isoforms in Aralast was suggested by the results of CZE, and was confirmed by IEF. CONCLUSIONS: Zemaira demonstrated greater purity compared with Aralast and Prolastin. Prolastin had more inactive A1-PI than Zemaira or Aralast. Isoform ratios appeared to be altered in Aralast. The results from this pilot study warrant further investigation.     

鸵鸟心脏细胞色素C的制备及性质

目的从鸵鸟心脏中分离提纯细胞色素C(cyt.c)并对其理化性质、活性进行考察。方法在酸性条件下从鸵鸟心脏中提取细胞色素C粗制品后 ,在离子交换色谱柱上进行纯化 ,用HPLC法、IEF法及SDS PAGE法检测所得细胞色素C纯度 ,用SDS PAGE法测定其分子质量 ,用IEF法测定其等电点。用药典方法进行含量测定、活力测定。结果经纯化后所得样品具有等电点均一性 ,达到了高效液相纯及SDS PAGE凝胶电泳纯。测得分子质量为 1 1 6kDa ,等电点为 1 0 1 0。单位重量鸵鸟心的细胞色素C产量为 92～ 1 47mg/kg,鸵鸟细胞色素C的平均活力为 81 5 0 %。结论鸵鸟心的细胞色素C含量丰富 ,用同样的工艺过程 ,单位重量原料的产量高于猪心 ,且有着比猪心细胞色素C更高的活性。     

Development and evaluation of a multiple-unit oral sustained release dosage form for S(+)-ibuprofen: preparation and release kinetics

This paper describes the conditions of preparation of poly(epsilon-caprolactone) (PCL) microparticles with a mean size between 5 and 10 microm, obtained by a double emulsion-solvent evaporation technique, suitable for oral vaccine delivery. Bovine serum albumin (BSA) was used as water-soluble model antigen for encapsulation. Different parameters influencing the microparticle size, the BSA loading and entrapment efficiency were investigated. Spherical, smooth and homogeneously distributed microparticles were produced with a BSA loading and entrapment efficiency reaching, respectively, 5% (w/w) and 30%. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) analyses of BSA released from these particles confirmed that the entrapped protein seemed to remain unaltered by the protein encapsulation process. Copyright     

PAGE在海洋酸性寡糖分析中的应用

报道了聚丙烯酰凝胶电泳（PAGE）法测定海洋酸性寡糖如卡拉胶,岩聚糖硫酸酯及褐藻胶及其衍生物的方法,实验结果表明：PAGE对海洋酸性寡糖具有较高的分离能力,电泳后的凝胶经阿利辛蓝（Alcian blue)染色并用扫描仪保存图象,所得图象通过专用分析软件对结果进行数字化分析,经和肝素寡糖标准品比较,可得到样品纯度及相对分子质量的分布信息。