Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

家蝇幼虫差异表达基因的克隆筛选与分析

目的是利用mRNA差异显示(DDRT-PCR)技术对诱导后家蝇幼虫差异表达基因进行克隆分析。取诱导和未诱导家蝇(Musca domestica vicina)三龄幼虫总RNA进行mRNA差异显示反应,产物经6％非变性聚丙烯酰胺凝胶电泳展开,银染分析后,回收差异显示条带。随机选取4条诱导家蝇三龄幼虫中上调表达的基因条带,进行Northern杂交验证,有2条被验证为真实带,命名为YD1和YD2。对这2条基因片段进行T-A克隆和测序,长度分别为495bp和265bp,登录NCBI运用Blastn程序进行同源性比较,发现两者与任何已知基因同源性都低于70％,提示为两个未报道的新基因,在家蝇免疫反应中可能起着一定的作用。     

Simplified riboprobe purification using translucent straws as gel tubes

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.     

Anomalous behavior of bacteriophage lambda polypeptides in polyacrylamide gels: resolution, identification, and control of the lambda rex gene product.

The resolution of lambia proteins was compared on the two types of sodium dodecyl sulfate-polyacrylamide gels commonly in use. The two kinds of gel differ essentially in the ratio of the cross-linker, N'-N-bismethylene-acrylamide (bisacrylamide), to acrylamide monomer. Several lambda proteins migrate relatively more slowly in gels with high bisacrylamide/acrylamide ratios (HB gels) than in gels with low ratios, although the two types of gel are of roughly equivalent porosity. This effect is illustrated by a change in relative position of both the Rex and Int proteins, with apparent increases in molecular weight of about 8 and 15%, respectively, in the HB gels. This work confirms that like repressor and Int, the 28.5-kilodalton protein, identified as Rex on HB gels, is postively regulated by the lambdacII and cIII products and negatively controlled Cro. An intact y site is required for Rex and repressor expression after infection, whereas their synthesis in a lysogen is dependent upon a functional maintenance promoter, Prm.     

In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs

A preliminary translational map of the Autographa californica genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected Spodoptera frugiperda cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of l-[(3)H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19 A. californica nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with l-[(3)H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.