Colocalization of the myc oncogene protein and small nuclear ribonucleoprotein particles.
Colocalization of the myc oncogene protein and small nuclear ribonucleoprotein particles.
复制标题
myc 癌基因蛋白和小核核糖核蛋白颗粒的共定位。
DOI：
10.1101/sqb.1986.051.01.107
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Spector,DL
中科院分区：
文献类型：
--
作者：
Sullivan,NF;Watt,RA;Delannoy,MR;Green,CL;Spector,DL
材料和方法鹌鹑Q8细胞或科洛320细胞在玻璃盖片上在含有10070胎牛血清的Dulbecco改良Eagle培养基（Gibco）中生长。c-myc（Watt等，1985）和Sm（Lerner等，1981; Spector和Smith 1986）的抗体先前已被鉴定。将在盖玻片上生长的细胞在甲醇中于-20 ° C固定1分钟，在磷酸盐缓冲盐水（PBS，pH 7.4）中冲洗，并与小鼠单克隆抗Sm抗体（1：6）和兔多克隆抗c-myc抗体（1：200）在加湿室中于20 ° C孵育30分钟。将细胞在几种变化的PBS中洗涤，并在FITC-2000中孵育。（山羊抗小鼠）和TRITC-（山羊抗兔）缀合的和亲和纯化的二抗，稀释度为1：在20 ℃下培养30分钟。数次PBS洗涤后，将盖玻片在Hoechst染色剂（1 μ g/ml）中培养30秒，在PBS中冲洗，并固定在含有没食子酸正丙酯的甘油/PBS中以抑制随后的光漂白。用配备落射荧光的Zeiss标准显微镜使用Neofluar（NA 1.30）100 x物镜检查细胞。
MATERIALS AND METHODSQuail Q8 cells or COLO 320 cells were grown on glass coverslips in Dulbecco's modified Eagle medium (Gibco) containing 10070 fetal calf serum. Antibodies to c-myc (Watt et al. 1985) and Sm (Lerner et al. 1981; Spector and Smith 1986) have been previously characterized. Cells grown on coverslips were fixed in methanol for 1 minute at-20~ rinsed in phosphate-buffered saline (PBS, pH 7.4), and incubated with mouse monoclonal anti-Sm antibody (1: 6) and rabbit polyclonal anti-c-myc antibody (1: 200) for 30 minutes at 20~ in a humidified chamber. Cells were washed in several changes of PBS and incubated in FITC-(goat-anti-mouse) and TRITC-(goat-anti-rabbit) conjugated and affinity-purified secondary antibodies at a dilution of 1: 20 for 30 minutes at 20~ After several PBS washes coverslips were incubated in Hoechst stain (1# g/ml) for 30 seconds, rinsed in PBS, and mounted in glycerol/PBS containing 3070 n-propyl gallate to inhibit subsequent photobleaching. Cells were examined with a Zeiss standard microscope equipped for epifluorescence using a Neofluar (NA 1.30) 100 x objective.