一种改良的蛋白质电泳考马斯亮蓝G-250染色方法

目的建立一种简便、低毒的蛋白质聚丙烯酰胺凝胶电泳染色方法。方法电泳后的凝胶经短暂固定后,用含氨基磺酸的考马斯亮蓝G-250溶液染色,无需脱色。结果该方法灵敏度高于经典考马斯亮蓝R-250染色方法,并去除了常规染色和脱色液中有毒的甲醇等溶剂。结论改良方法具有灵敏度高、染色背景浅、节省染料、直接显色无需脱色及配方安全低毒等优点。     

考马期亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人检测人精子顶体反应的简便实用的新技术，用３．５％高氧酸水溶液本制０．０５％考马斯亮蓝染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不梁。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。     

核内M-CSF对NIH3T3细胞内微丝的影响

目的建立M-CSF核内稳定表达的NIH3T3细胞系,探讨核内M-CSF对细胞骨架的影响。方法经脂质体介导核内定位表达重组体pCMV/M-CSF转染NIH3T3细胞,G418筛选后,用Rt-PCR、免疫细胞化学鉴定其在真核细胞中的表达及定位分布,用考马斯亮蓝染色细胞微丝测定M-CSF进入细胞核后对细胞骨架的影响。结果RT-PCR结果显示转染pCMV/M-CSF的NIH3T3细胞能稳定表达M-CSFmRNA;免疫细胞化学结果显示表达的M-CSF定位于NIH3T3细胞核。考马斯亮蓝染色结果显示转染pCMV/M-CSF的NIH3T3细胞内微丝数量减少,排列紊乱。结论核内M-CSF可引起NIH3T3细胞内微丝数量减少,排列紊乱。     

考马斯亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人精子顶体反应的简便实用的新技术，用３．５％高氯酸水溶液配制０．０５％考马斯亮蓝（Ｒ２５０）染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不染。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。方法学检验证明新技术结果可靠。本法操作简便、经济，普通显微镜即可检测，易于推广，克服了原有技术复杂昂贵的缺点，具有研究和临床诊断的良好价值。     

研磨珠均质仪提取增生性瘢痕组织蛋白质的方法

目的 利用研磨珠均质仪,从临床增生性瘢痕组织样本中充分提取蛋白质。方法 临床收集烧伤后增生性瘢痕组织样本,通过玻璃匀浆器、研磨珠均质仪等方法,提取样本总蛋白,比较提取后残渣,BCA测定总蛋白浓度,蛋白凝胶电泳考马斯亮蓝染色观察蛋白质条带完整性,和western blot比较前列腺素D合成酶的含量（PTGDS）。结果 玻璃匀浆器与研磨珠均质仪联合方法残渣呈絮状均匀,获得蛋白质量最大,差异具有统计学意义（P＜0.05）,蛋白凝胶电泳考马斯亮蓝染色样本均未降解,Western blot测得联合方法提取样本中前列腺素D合成酶含量更高。结论 玻璃匀浆器与研磨珠均质仪的联合提取临床增生性瘢痕组织样本中获得蛋白质较为高效,是一种临床瘢痕组织蛋白样本提取方法。     

人穿孔素在sf9昆虫细胞中的表达和纯化

目的建立重组人穿孔素的表达和快速纯化方法。方法在sf9昆虫细胞内表达带His标签人穿孔素。使用人工制作简易的镍(Ni)柱装置进行蛋白质亲和层析。采用考马斯亮蓝染色、银染法及流式细胞计量术纯化的蛋白分析其理化性质;用人乳腺癌细胞MCF-7的PI染色法鉴定其生物学活性。结果纯化出的穿孔素具有在细胞膜打孔的生物学活性。与常规蛋白纯化方法如离子交换色谱、分子筛和疏水作用层析等方法相比,该方法操作简便、成本低,为人穿孔素接下来的研究以及其他蛋白质的纯化提供一定参考。结论以一种简单的方法在sf9昆虫细胞内表达并纯化出了有活性的人穿孔素。     

间接ELISA法测定大肠杆菌DH5α菌体蛋白含量的实验研究

采用大肠杆菌ＤＨ５α菌体蛋白免疫家兔制备抗血清，建立了间接ＥＬＩＳＡ方法，经初步测定，该方法的灵敏度为０．５ｎｇ／ｍｌ，比聚丙烯酰胺凝胶电泳的考马斯亮蓝染色方法的灵敏度高３０００倍，比银染色方法的灵敏度高１５０倍。在２０－６２０ｎｇ／ｍｌ范围内测定呈直线关系，直线相关系数为０．８９５。     

利用考马斯亮蓝显示精母细胞性泡的轴成分

已证明哺乳动物的精母细胞的早前期中,X和Y染色体形成一个致密的团块,称为性泡。近来,作者发展了一种考马斯亮蓝(CBB)染色技术,用此法可清晰地看到有功能的NOR,并发现同一技术也能使大鼠和小鼠精母细胞的性泡里的性染色体轴成分着色。     

连结蛋白36和闭锁小带蛋白1的PDZ结构域关系

目的:探讨连结蛋白36(Cx36)和闭锁小带蛋白1(ZO-1)相互作用的PDZ结构域。方法:构建pGEX-3X GST-PDZ重组载体在DH5α细菌表达,加IPTG产生融合蛋白,经纯化,免疫印迹分析Cx36与ZO-1相互作用的PDZ结构域,PVDF膜洗脱后抗GST和考马斯亮蓝染色。结果:构建了pGEX-3X GST-PDZ重组载体,与转染Cx36的HeLa细胞孵育,免疫印迹显示Cx36与ZO-1的PDZ1结构域结合。而对照组Cx43与ZO-1的PDZ2结构域结合。实验组在相对分子质量为40 000-42 000有蛋白带,提示GST-PDZ融合蛋白表达。与鼠视网膜组织孵育免疫印迹显示Cx36与ZO-1的PDZ1结构域结合,而对照组Cx43与ZO-1的PDZ2结构域结合,相同膜洗脱后抗GST抗体和考马斯亮蓝染色显示GST-PDZ融合蛋白表达。结论:Cx36与ZO-1的PDZ1结构域结合。     

SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

目的　通过昆虫杆状病毒表达系统表达SETD4（SET domain-containing 4）蛋白,并纯化SETD4蛋白,为深入探讨SETD4的功能奠定基础。 方法　提取小鼠正常肝组织的RNA,通过RT-PCR扩增SETD4基因,并克隆至pFastBac-HTB构建重组载体,再转座获得重组杆粒;通过脂质体介导将重组杆粒转染SF9细胞产生重组病毒,扩增病毒感染细胞并获得重组蛋白;利用Ni2+亲和柱来纯化蛋白,并通过Western Blot及考马斯亮蓝染色鉴定SETD4蛋白。 结果　经双酶切鉴定及测序证实SETD4基因插入了供体质粒;经PCR鉴定证实SETD4基因插入了穿梭载体;经考马斯亮蓝染色证实纯化得到重组蛋白,用His-Tag抗体和SETD4特异性抗体在50 kD处可检测到目的条带。 结论　成功利用昆虫杆状病毒表达系统够表达了SETD4,并纯化了SETD4。     

Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS-PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107,000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol-diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445-480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M(r) 260,000) after silver staining and Coomassie staining of 4-15% gradient SDS-PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS-PAGE bands and the apo B-48 in a protein range of 0-3 microg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.     

Coomassie brilliant blue R-250 as a highly sensitive pre-stain for immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis and raising monospecific antibodies

We hereby describe electrophoretic analysis of normal human serum pre-stained with Coomassie Brilliant Blue R-250 (CBB). The proposed method was optimized by studying as many as 450 disc electrophoretic separations and 30 variables. The method when compared with the post-electrophoretic staining by Amido Black (AB) revealed that the pre-stained discs were intensely well defined and resolved within 2 hours with a transparent gel. Gels stained with AB, despite a prolonged destaining, showed a residual dye retention making identification of the faint components difficult. Protein eluted from the CBB pre-stained gels retained its purity and immunoreactivity and conjugates of the two prototype proteins namely the albumin and the transferrin produced high-titre monospecific antisera in immunized rabbits.     

Electrophoretic analysis of erythrocyte membrane proteins and glycoproteins from different species

The erythrocyte membrane proteins and glycoproteins of man, rat, mouse, sheep and dog were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using a discontinuous buffer system. Considerable similarities between the species were observed in the pattern of protein bands seen when gels were stained with Coomassie Blue. Equivalents to human Bands 1, 2, 3, 4.2, 5 and 8 appeared to be present in the rat, mouse, sheep and dog, and Band 4.1 was identified as a closely spaced doublet in all species except the rat.When RBC membranes were stained with periodic acid-Schiff (PAS) after SDS-PAGE analysis, glycoproteins equivalent to human glycophorins were identified in all the species studied. However, in contrast to the overall similarity of the protein patterns, the number, relative staining intensity and apparent molecular masses of the PAS-stained bands differed between species.The silver stain was assessed in the detection of RBC membrane proteins in polyacrylamide gels, and found to be more sensitive than Coomassie Blue. The technique also stained many of the PAS-positive glycophorins as diffuse orange zones, which could be distinguished from the darker protein bands by their differential colouration.In view of the interspecies variation in the glycophorins after SDS-PAGE, it is suggested that, unlike the membrane proteins, their functions do not require a conserved structure.     

食入性过敏原-杏仁蛋白组分的双向电泳分析

目的 :应用蛋白质双向电泳的分析技术 ,对一种主要的食入性过敏原杏仁果实的蛋白质组分进行分析。方法 :通过三氯乙酸法提取杏仁果实总蛋白质 ,通过等电聚焦和第 2向SDS PAGE分析获得完整的杏仁果实全蛋白质的图谱 ,并应用相应分析软件 (ImageMaster 2D)对电泳图谱进行分析。结果 :通过等电聚焦和第 2向SDS PAGE ,有 188个不同的蛋白质组分被检测出来。其中大约 2 8%的蛋白质等电点 (pI)在 4 .5～ 5 .5之间 ,大约 6 2 %的蛋白质的相对分子质量 (Mr)在 (2 0～ 2 5 )× 10 3 之间。结论 :所获得的高分辨率的双向电泳图谱 ,是我国杏仁果实蛋白质第一张完整的蛋白质图谱 ,为今后杏仁果实中致敏蛋白质的检测 ,分离、基因克隆和变应原的标准化奠定了基础。     

Effects of low and high relative molecular protein mass on four methods for total protein determination in urine.

Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.     

Comparison of protein and lipopolysaccharide patterns of several Coxiella burnetii strains in phase I

Electrophoresis in polyacrylamide gel gradients in the presence of sodium dodecyl sulphate revealed at least 18 identical protein bands in eight strains of Coxiella burnetii. By staining with Coomassie Brilliant Blue R-250 it was possible to visualize clearly at least 40 proteins. The protein pattern showed the greatest variability in the region from 27 to 18.5 kD. Strains S and Priscilla differed from the other strains also in proteins smaller than 18.5 kD. The lipopolysaccharide pattern obtained by the same technique consisted of from 6 to 12 various bands in the region from 23.5 to 11.8 kD. The protein and lipopolysaccharide patterns of four strains isolated from ticks did not significantly differ from those of strains Henzerling and L-35 isolated from men.     

Proteinuria after renal transplantation: Diagnosis with highly sensitive silver stain in sodium dodecylsulphate-polyacrylamide gradient gel electrophoresis

Summary Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of urinary proteins is becoming increasingly significant in monitoring renal allograft recipients. After conventional Coomassie Blue staining, changes in renal proteinuric patterns during acute renal transplant rejection could not be seen in many cases. Discrimination between rejection and nephrotoxic side effects of cyclosporine overdosage was also not possible. We therefore examined the possible advantages of a highly sensitive silver staining technique in this study. A total of 734 urine samples obtained from 38 patients after allogenic kidney transplantation were examined by SDS-PAGE with consecutive Coomassie Blue and silver staining. Twenty-two histologically proven rejections and 20 cyclosporine overdosage episodes were diagnosed in these patients within a time period of up to 524 days after transplantation. No changes in proteinuric patterns were seen in 9 of 22 patients after Coomassie stain during rejection, and only 12 cases showed a rise of glomerular protein bands, whereas silver stain revealed an increase in 19 of 22 cases. Discrimination between cyclosporine overdosage and rejection was possible with a probability ofp<0.001 after silver stain when using the changes in the number of glomerular protein bands as a criterion. These findings suggest that application of a highly sensitive silver stain instead of the conventional Coomassie stain after SDS-PAGE reflects considerable progress in monitoring renal allograft recipients.Abbreviation SDS-PAGE Sodium dodecylsulphate-polyacrylamide gel electrophoresis     

rBla g 2变应原的PEG修饰与免疫学特性分析

目的 研究化学修饰剂聚乙二醇(PEG)对德国小蠊(蟑螂)重组变应原rBla g 2变应原性的影响.方法 rBla g2在大肠肝菌中表达后,采用Ni+亲和层析纯化,然后甲氧基聚乙二醇2-N-羟基琥珀酰亚胺酯(mPEG2-NHS,Mr为10×103)修饰,经阳离子交换层析纯化,用SDS-PAGE、免疫印迹及ELISA分析其生物学特性.结果 rBla g 2纯化后的相对分子质量(Mr)约39×103;sDs-PAGE分析PEG-rBla g 2修饰产物,考马斯亮蓝染色可呈现5条带,而I2-KI染色则呈现7条带.对修饰产物的纯化表明阳离子交换层析可分离rBla g 2和PEG-rBla g 2;对修饰产物的免疫印迹分析显示Mr为100×103和130×103的修饰带可与蟑螂过敏患者血清特异性IgE结合,同时ELISA分析结果表明PEG-rBla g2复合物的体外免疫学反应性显著下降,仅为原来的42%.结论 PEG修饰可保持rB-la g 2与特异性抗体结合能力,却大大降低变应原的体外免疫学反应性,为重组低致敏原的研究提供了基础资料.     

Evaluation of allergenicity of the extract of Japanese juniper pollen reacting to sera from asthmatic children--by the methods of enzyme-linked immunosorbent assay and immunoblotting]

We investigated the allergenicity of pollen extract of Japanese juniper (Juniperus rigida, Cupressaceae family) to sera of 49 asthmatic children by the methods of enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Three bands stained with Coomassie Brilliant Blue R were detected on SDS-PAGE. Sera from 27 (55.1%) out of the 49 children showed positive reaction to the pollen extract in ELISA. The same sera from the 27 children were used as the first antibody in immunoblotting, which confirmed the presence of a band of protein with 70 K dalton molecular weight (M.W) common to the all sera. This band was bound with concanavalin A in lectins. We successfully purified the antigenic substance of Japanese juniper pollen from this band by the electroelution method. The major allergen of Japanese juniper pollen is glycoprotein with 70 KM.W. On sandwich-ELISA, there was no reaction of Sugi Basic Protein (SBP) and anti-SBP to Japanese juniper is an allergen that has no cross-reactivity with SBP.