一种简易的聚丙烯酰胺凝胶干胶制作法

在聚丙烯酰胺凝胶电泳中经常遇到凝胶干胶制作不好的问题,即使在有干胶机的实验室也常发生凝胶裂开、皱缩、电泳条带扩散、干胶质量不理想的情况,一些文献介绍的方法也常发生类似问题。我们在干胶的方法上作了一些摸索和改进,总结出一种简易的、     

一种新的制备酸性聚丙烯酰胺凝胶的引发系统

一种新的制备酸性聚丙烯酰胺凝胶的引发系统孙新立＊孙海虹王友爱（河北师范大学生物系,石家庄０５００１６）关键词引发系统;转化率;聚合;酸性凝胶;成胶时间碱性聚丙烯酰胺凝胶电泳已经广泛应用于生化分析的各个领域。但酸性聚丙烯酰胺凝胶电泳、过硫酸铵（ＡＰ）－...     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

聚丙烯酰胺凝胶电泳的一种辅助辨型方法

聚丙烯酰胺凝胶电泳(PAGE)是实验室常用的基本鉴定方法之一.但由于做胶方式以及各实验室仪器设备良莠不齐,经过一系列纷繁复杂的操作,电泳结果未必尽如人意.其中凝胶带型的判断就是一个较为困惑的问题,一般的聚丙烯酰胺胶跑出的条带总体上都有弧度,越是靠近凝胶两侧越是严重.在本实验中,我们设计了一种较为简单易行的方案,即将难以区分的条带样品做成混合样marker来辅助辨型,通过调整电压和凝胶浓度来达到区分条带的目的.我们用该方案可以将相差0～3 bp大小的条带区分开来.     

细菌蛋白的聚丙烯酰胺凝胶电泳鉴定

细菌蛋白的聚丙烯酰胺凝胶电泳鉴定华西医科大学口腔医学院口腔生物医学工程重点实验室成都６１００４１黄毅早在１９０８年,电泳现象就已被发现,Ｔｉｓｅｌｉｕｓ于１９３７年对电泳仪器作了重大改进,随后电泳技术才日益普及并成为鉴定生物大分子的基本工具。Ｆｏｗｌ...     

检测聚丙烯酰胺凝胶中蛋白质的3种染色方法比较

用考马斯亮蓝染色、银染色、铜染色等 3种方法对同一种蛋白质染色的灵敏度、快速性进行比较 ,得出 3种蛋白质染色方法的优缺点 ,为蛋白质电泳染色合理选用不同方法提供依据     

一种简便的凝胶干燥法

SDS-PAGE(聚丙烯酰胺凝胶电泳)已成为分析蛋白质核酸等样品的一种常用方法。为使染色后的凝胶长期保存,需制成干胶。下面介绍一种比已报道的更为简便易行的凝胶干燥方法。取一块如电泳用大小的玻璃扳,将二张略大于玻璃板的玻璃纸浸于脱色液中。先取一张玻璃纸平铺于玻璃板上,从一端向另一端平铺,抚平去气泡,涂一层50%甘油。将凝胶放其上,在凝胶面上也涂一层50%甘油。将另一张玻璃纸覆盖在凝胶上从一端开始慢慢平铺,抚平赶尽气泡,将大于玻璃板的玻璃纸向玻璃板     

改良聚丙烯酰胺凝胶电泳检测DNA

本文介绍了应用聚丙烯酰胺凝胶电泳检测DNA的改进方法。与以往方法相比,本方法从配制凝胶储备液,无需封口的水平灌胶,适当地提高电泳的电压梯度,采用改良银染法检测,省却凝胶固定,银染法DNA的纯化方法,及溴化乙锭染色法的操作等一系列改进措施,使聚丙烯酰胺凝胶电泳检测DNA的操作得到简化,更便捷,并减小了对人与环境的毒害作用。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究*

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

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     

枸杞花药蛋白质组双向电泳体系的建立及应用

采用改良TCA丙酮沉淀结合Tris-HCl法提取枸杞花药蛋白质,对蛋白质裂解液成分、IPG胶条的pH范围、上样量及染色方法进行了探索.结果表明:(1)采用17 cm胶条、400 μg的上样量、含有2 mol/L硫脲的裂解液,硝酸银染色,可得到重复性好、质量高的枸杞花药蛋白2-DE图谱,枸杞花药蛋白主要集中在pH 4～7范围.(2)采用该体系分析了‘宁杞1号’和‘宁杞5号’四分体时期花药蛋白,并利用PDQuest 8.0软件在pH 4～7的2DE图谱上检测到500多个蛋白点,其中差异表达量大于2倍的蛋白有25个.     

应激心肌细胞蛋白质组双向凝胶电泳分析

采用双向凝胶电泳技术和计算机辅助的图像分析方法 ,对去甲肾上腺素诱导的应激心肌细胞与正常心肌细胞蛋白质进行分离和比较分析 .正常心肌细胞可分离 12 32± 5 6个蛋白点 ,蛋白点匹配率为 83 3%± 1 0 %.有 11种蛋白质在NE应激后发生了明显和稳定的质和量的改变 (P <0 0 5 ) ,其中 6种 (Mr pI :4 9 7kD 7 8,38 3kD 5 9,37 1kD 6 6 ,2 9 3kD 7 4 ,18 7kD 6 1,18 5kD 7 7)在应激后表达降低 ,4种 (Mr pI:4 7 6kD 5 5 ,31 9kD 4 4 ,2 6 6kD 4 6 ,33 2kD 8 1)在应激后表达增高 ,1种 (Mr pI:19 4kD 6 9)只在应激后发生表达 .这些差异表达的蛋白质可能参与了心血管应激反应乃至应激损伤发生的过程 .     

双向电泳分析鸢尾绿白嵌合叶片的蛋白质

利用双向聚丙烯酰胺凝胶电泳对鸢尾（Iris japonica)绿白嵌合叶片的蛋白质进行分离,并初步鉴定了蛋白质的相对分子量和等电点。每个电泳图谱共检测到400余个蛋白点,其中至少13个蛋白的表达变化明显;结果表明,嵌合叶片的绿色与白色叶组织具有明显不同的蛋白质双向电泳图谱。与数据库中拟南芥双向电泳图谱相比较,发现Rubisco大亚基,标记为W和T蛋白的表达变化与产生绿白嵌合叶片的表型密切相关。     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Assembly of the cytochrome bo3 complex

An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo(3) of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.     

聚丙烯酰胺凝胶碎块吸收洗脱法浓缩病毒的实验研究及应用

本文报告聚丙烯酰胺凝胶(PAG)碎块吸收洗脱法在浓缩新城鸡瘟病毒(NDV)和人轮状病毒(HRV)方面的实验研究及应用。我们通过一次和二次浓缩,分别将NDV感染的鸡胚尿液稀释液浓缩了12.1倍和90.2倍,血凝滴度相应增高,总病毒回收率分别达到90.2％84.9％。用二次浓缩后的含HRV粪便上清液进行RNA PAGE银染色检测,获得了HRV RNA电泳型检测阳性稀释度提高32倍左右的结果。据此,采用这一方法,对临床RNA PAGE检测阴性的33例婴幼儿腹泻粪便进行PAG浓缩处理后再检测,发现有12例转为阳性,阳转率为36.4％。从而,使HRV总检出率由67％增至79％。     

A simple technique for the preparation and storage of sucrose gradients

A method for preparing multiple sucrose gradients by quickly freezing layers of sucrose has been developed. These gradients may be stored in the freezer indefinitely, and thawed from 8 to 24 h at 4 degrees C before use. The middle region of the resulting sucrose gradients was linear. Thawing time and centrifugation had little effect on the shape of the gradient. The method is applicable for both small- and large-volume centrifuge tubes. Gradients prepared in the same batch were nearly identical.     

基于非变性聚丙烯酰胺凝胶电泳的菜心SCoT分析

以菜心(Brassica rapa var. parachinensis)抗小菜蛾品种Caixin65和感小菜蛾品种Caixin69及6份F2株系为材料,利用非变性聚丙烯酰胺凝胶电泳检测菜心抗虫株系的SCoT多态性,同时进行SCoT多态性的非变性聚丙烯酰胺凝胶电泳检测效率、遗传多样性分析和差异条带克隆分析。结果表明,非变性聚丙烯酰胺凝胶能检测出亲本与子代间的SCoT多态性和遗传多样性变化,条带数量较多且清晰,提高了SCoT标记检测效率。选取亲本的10条非变性聚丙烯酰胺凝胶差异片段克隆测序,获得感虫序列4条、抗虫序列6条,GENSCAN预测其中8条具有启动子、终止子、阅读框等基因结构序列。同源性检索分析表明,感虫序列分别与泛素羧基末端水解酶mRNA序列、大白菜克隆序列、白菜线粒体丙酮酸载体蛋白序列、甘蓝基因组编码未知蛋白的HDEM序列同源,抗虫序列分别与白菜RNA假尿苷合酶4线粒体mRNA序列、京水菜线粒体DNA序列、白菜未知蛋白mRNA序列、白菜4-香豆酸：辅酶A连接酶mRNA序列、大白菜克隆序列、哈茨木霉mRNA序列同源。本研究提高了SCoT标记清晰度、遗传多样性检测水平和差异片段克隆的精准性,使得SCoT成为批量克隆差异片段的高效工具,有助于挖掘SCoT功能性标记信息,开展初步的功能基因组学研究,提高优异株系筛选鉴定效率,加快育种进程,为菜心抗虫性机制的进一步研究提供理论基础。     

Electrophoresis in the presence of Coomassie brilliant blue R-250 stains polyacrylamide gels during protein fractionation

A method of staining polyacrylamide gels in which the dye is electrophoresed together with the sample is proposed. The method cuts short and simplifies the conventional electrophoresis procedure by eliminating the separate poststaining step. In the gels run in the presence of sodium dodecyl sulfate, the method produces protein staining patterns which are quantitatively identical to the ones obtained by conventional staining procedure. Additional advantages of the method are easy control over the degree of staining and homogenous staining independent of the gel thickness and concentration of the dye.     

有和无甘油的聚丙烯酰胺胶在检测突变时的差别

有文献报道在非变性的聚丙烯酰胺中加入甘油可提高SSCP检测的灵敏度。我们的实验结果建议研究者在进行SSCP筛查未知突变时最好采用不加甘油的非变性的聚丙烯酰胺胶,这既省力省钱,又灵敏。在判读SSCP胶时,千万不要看到在双链带位置有一条比正常迁移率慢的带就判定为插入突变。此时要判定突变的性质,最好测序。 Abstract：It was reported that glycerol in the non-denatured SSCP polyacrylamide gel could increase the sensibility of detecting mutation. We detected the mutation of PKD 1 gene in the patients with autosomal dominant polycystic kidney disease.PCR com bined with SSCP(single-strained conformation polymorphism),the non-denatured 10% polyacrylamide gel without glycerol or 10% polyacrylamide gel with 5% glycerol and DNA sequencing method were used.Our results showed that four single strand b ands were found in the non-denatured polyacrylamide gel without glycerol while t wo single strand bands were found in the polyacrylamide gel with glycerol in the same patient.Sequence showed there is a deletion of G in one DNA molecular and a G→A substitution in another DNA molecular in the patient with abnormal shift SSCP bands.Therefore, our experiment suggested that non-denat ured polyacrylamide gel was better than the polyacrylamide gel with glycerol in detection mutation,and it will save labor and money.It also suggeste d that one basedeletion can cause a slow double-strand DNA following the normal double strand band,which was caused by the heterogeneous DNA molecule formed bet ween the normal DNA strand and the one base deletion DNA strand with the protrud ing base.Our results suggest that when judging mutation in SSCP gel,it is not re liable to decide that mutation is inversion according to slow mobility in the ge l,and when the characteristic of mutation need to be judged,it must be sequenced .