盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验的几点改进

对盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验提出了几点改进,以满足本科生实验的要求。实验主要比较和分析了两种封胶方法（原胶布封胶与改进的琼脂糖封胶）和两种染色方法（原考马斯亮蓝染色法与改进的考马斯亮蓝染色法）对凝胶分离血清蛋白实验的影响。结果显示,改进的盘状聚丙烯酰胺凝胶电泳法是一种灵敏、快速、简便、安全、分辨率高的实验方法。结论：改进的盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验非常适合本科生实验。     

蛙胚胎发育过程的扫描电镜照片说明

封二,封三刊登的是一组蛙(Rana pipiens)胚发育过程的扫描电镜照片。蛙的胚胎发育,从卵裂到发育成早期蝌蚪,都是在受精膜和胶被膜中进行的。最后孵出时,卵膜和胶被膜被胚胎分泌的酶所溶解,胚自胶被膜中出,不久便能游动。在这一组照片中,除了图1中的未受精的卵是只去掉了胶被膜外,其余的都是除去了胶被膜和受精膜以后的情况。图1表示的是一未受精的卵。在以后受精的时候,精子进入动物半球,约一小时后,在精子进入点的对侧,动物半球与植物半球的交界处,出现灰色新月(grey crcscent)。     

塔拉豆中塔拉胶含量的分析测定方法研究

介绍了塔拉豆的研究现状,并对塔拉豆中塔拉胶含量的分析测定方法进行了研究。结果显示:塔拉胶的分离条件为:烘炒温度为150℃,时间7 min,每小组为4颗成熟饱满的塔拉豆。烘炒后破碎,塔拉胶和其余两部分的分离效果好,塔拉胶的经济性状也完整。每个分析样品所需塔拉豆的数量:5小组(即20颗塔拉豆)为一组实验结果,三组平行实验结果经过方差分析达到实验要求。     

假酸浆子胶与黄原胶混胶流变性研究

实验研究了放置温度、时间、冻融、pH、盐以及柠檬酸对黄原胶和假酸浆子胶混胶黏度的影响.结果表明:黄原胶和假酸浆子胶有协效性,当假酸浆子胶与黄原胶的质量比15:85时,二者的协同增效性最高,胶溶液为非牛顿型流体,且变化满足Herschel-Bulkley方程.温度、时间对混胶有一定的影响.进一步对其研究表明:冻融、pH、盐以及柠檬酸都对其影响较小.     

优化胶粘贴法建立裸鼠胃癌原位种植模型

目的通过对两种胶粘贴法建立的胃癌原位种植动物模型的比较研究,为探讨胃癌的发病机制和实验治疗提供理想的动物模型。方法用OB胶和FS生物蛋白胶法分别建立胃癌原位种植动物模型,观察和比较两种方法所建立的模型肿瘤生长状况、转移情况和形态学变化。结果FS生物蛋白胶组未出现肿瘤大片坏死,腹水形成率为85.7%,幽门梗阻发生率为57.1%;而OB胶组肿瘤大片坏死发生率为100%,腹水形成率为14.3%,未出现幽门梗阻。FS生物蛋白胶组有三例出现了肺和脑转移。结论FS生物蛋白胶法建立的裸鼠胃癌原位种植动物模型能更好的模拟人胃癌患者的临床过程,为研究人胃癌转移机制和实验治疗提供理想的动物模型。     

改性黄原胶酯化条件的优化及结构的表征

为了提高黄原胶的速溶性和粘度,将黄原胶进行改性处理。将黄原胶与马来酸酐进行酯化反应,探讨了黄原胶与马来酸酐摩尔比、反应时间和反应温度等因素的影响,以取代度为指标,利用响应面方法确定,该酯化反应的最优条件为:黄原胶与马来酸酐摩尔比1∶11.5、反应时间24.4 h、反应温度66℃。对改性黄原胶进行红外光谱、光散射和X-射线衍射等结构表征,表明酯化改性成功,且进一步解释了速溶性和粘度提高的原因。改性黄原胶细胞毒性实验,显示无毒性。结果表明,改性黄原胶的速溶性和粘度有很大提高,0.2%改性黄原胶的速溶性和粘度较对照提高了近3倍,在食品、药品等领域具有潜在的应用价值。     

黄原胶（Xanthan Gum）对小鼠免疫功能的影响

给小鼠每天分别灌胃10,2和0．4μg/g的黄原胶溶液14d,研究观察黄原胶对小鼠脾脏和胸腺、IgM－PFC反对小鼠迟发型变态反应（DTH）的影响。实验发现,黄原胶对小鼠脾脏和胸腺增重及DTH的影响未见明显变化,而使小鼠的IgM-PFC显著增加。实验结果表明,黄原胶对增加小鼠脾脏和胸腺重量及增强小鼠细胞免疫功能作用不大,而对小鼠的体液免疫功能具有明显的增强作用。     

植物多糖胶流变性质的研究

胡芦巴、皂荚、野皂荚和塔拉多糖胶水溶液随着浓度的增加,溶液表现为假塑性流体．表现粘度随剪切速率增大而减小。通过Cross模型和实验数据计算得到1％多糖胶零剪切速率时表现粘度η0、无穷大剪切速率时表现粘度η∞和弹性松驰时间等重要流变参数。根据Arrhenius方程和实验数据计算得到1％浓度多糖胶的粘度流化能E和常数A,皂荚胶和塔拉胶的粘流活化能大。时间对多糖胶液粘度影响较大,新配制的多糖胶液随着水化时间的延长,胶液表观粘度增大,胶液放置时间超过24h后,表现粘度随时间的延长而降低。pH值、冻融处理和盐离子对多糖胶表现粘度的影响不太大。多糖胶与硼离子形成的冻胶．其粘度因多糖胶品种而异,其中胡芦巴冻胶粘度最高,是野皂荚冻胶粘度的165倍和瓜尔胶冻胶粘度的2．7倍。     

如何正确估计蛋白质SDS聚丙烯酰胺凝胶电泳的样品加样量

在做蛋白质的SDS聚丙烯酰胺凝胶电泳（SDS—PAGE）时,使用的仪器设备、做胶和制样的方法都差不多,但结果却五花八门,相去甚远。有的条带清新,背景干净,令人赏心悦目;有的却条带模糊、肥瘦不均,其貌不扬;有的甚至歪歪扭扭,画成了地图。看来并不困难的操作,结果为什么会有如此之大的区别？除了试剂质量、制胶过程和跑胶的具体细节外。失败的主要原因往往在于样品制备的缺陷和加样量不适当。只有在样品制备良好、加样量又适当的情况下,     

使用胰蛋白酶进行胶内酶解

在蛋白质组学实验中,双向电泳分离得到的蛋白质点通常需要进行胶内酶解后再使用质谱鉴定,而胶内酶解是否成功直接决定了质谱鉴定的结果。本文以最常用的胰蛋白酶为例讲述胶内酶解的具体操作。     

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.     

聚皂凝胶对蛋白分子的吸附与释放

以牛血清白蛋白为对象,研究了丙烯酸－丙烯酰胺型９ＰＢｕＡＭＡ）和疏水改性的四乙烯五胺－环（ＨＰＴＥ）聚合物凝胶对蛋白质的及附与释放过程。在吸附过程中,当溶液ＰＨ值在牛血清白蛋白电点（４．７）附近时,凝胶蛋白抽的吸附值最大;而在释放过程中,ＰＨ＝７的释放率远大于其它ＰＨ值时的释放率。实验表明凝胶结构对蛋白质吸附与释放有较大影响。未疏水改性的凝胶与疏水改性的凝胶相比,前 的释放速率约为后者的２倍。结果     

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.     

An apparatus for the simultaneous casting of large numbers of uniform cylindrical polyacrylamide gels

An apparatus for the simultaneous casting of a large number of cylindrical polyacrylamide gels is described. Gels can be cast that are uniform with respect to length, loading-surface flatness, and internal polymerization properties. The basis of the method is casting the gels as an inverted single block which totally excludes oxygen from gel-loading surfaces during polymerization.     

Influence of gel dimensions on resolution and sample throughput on two-dimensional gels

To achieve high throughput and economical format of 2-D PAGE, comparison between gel size and resolution was conducted on human breast carcinoma cell line (MCF-7/AZ) proteins. SDS gel length showed a weaker influence of separation length on resolution in the second dimension, and there was little benefit of separation distances greater than 15 to 19 cm. IPG strip separation distances were very important with dramatic increase in resolution of longer gels compared with smaller gels, and maximal resolution was obtained using 18- and 24-cm IPG strips. Loading optimal amount of proteins on 2-D gels can also increase the number of detected spots. Therefore, taken together, compromise 2-D gels are crucial for higher capacity and higher throughput.     

A simple and reliable method for the drying of polyacrylamide slab gels

A simple method for the drying of polyacrylamide slab gels is described. 2-mm thick gels with gradients of 5–20% acrylamide dry without complications. The dried gels are transparent permitting transmission densitometry and fluorography.     

Inheritance of amylases in blood serum of cattle

Serum amylase variants are demonstrated by means of starch gel and polyacrylamide gel electrophoresis. Phenotypes, allele frequencies, and segregation data for Am₁ and Am₂ in the cattle breed Deutsche Schwarzbunte are given. Demonstration of Am₂ amylases was better in polyacrylamide gels and more isoenzymes were identified than in starch gels. The variants of Am₁ amylases found in STAGE could not be reproduced in PAGE by means of the described methods. Both enzyme systems seem to be profoundly different in their molecular constitution and action. For the animals, these differences could be of advantage in the adaption to external influences.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.     

Cell proliferation and cell sheet detachment from the positively and negatively charged nanocomposite hydrogels

The charged nanocomposite hydrogels (NC gels) were synthesized by copolymerization of positively or negatively chargeable monomer with N‐isopropylacrylamide (NIPAm) in the aqueous suspension of hectorite clay. The ionic NC gels preserved the thermo‐responsibility with the phase‐transition temperature below 37°C. The L929 cell proliferation was sensitive to charge polarity and charge density. As compared to the PNIPAm NC gel, the cationic NC gels with <5 mol % of 2‐(dimethylamino)ethyl methacrylate (DMAEMA) showed improved cell proliferation, whereas the cells grew slowly on the gels with negatively charged 2‐acrylamido‐2‐methylpropane sulfonic acid (AMPSNa). By lowering temperature, rapid cell sheet detachment was observed from the surface of ionic NC gels with 1 mol % of ionizable monomers. However, lager amount of AMPSNa or DMAEMA did not support rapid cell sheet detachment, probably owing to the adverse swelling effects and/or enhanced electrostatic attraction. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 58–65, 2014.