导演: 强·卡萨 编剧: Brad Mirman 主演: 黛米·摩尔 / 基弗·萨瑟兰 / 唐纳德·萨瑟兰 类型: 剧情 / 西部 制片国家/地区: 加拿大 / 法国 语言: 英语 上映日期: 2016-02-19 片长: 90分钟 IMDb链接: tt2271563 Kiefer and Donald Sutherland share the screen in this brooding western about an embittered gunslinger who attempts to make amends with his estranged father whilst their community is besieged by ruthless land-grabbers. 下载地址： http://page92.ctfile.com/fs/YHO146824663 http://www.yimuhe.com/file-3017430.html
导演: 强·卡萨 编剧: Brad Mirman 主演: 黛米·摩尔 / 基弗·萨瑟兰 / 唐纳德·萨瑟兰 类型: 剧情 / 西部 制片国家/地区: 加拿大 / 法国 语言: 英语 上映日期: 2016-02-19 片长: 1h 30min IMDb链接: tt2271563 Kiefer and Donald Sutherland share the screen in this brooding western about an embittered gunslinger who attempts to make amends with his estranged father whilst their community is besieged by ruthless land-grabbers. 下载地址： http://page92.ctfile.com/fs/8kl145908448 http://www.yimuhe.com/file-3010869.html
纳米粒子瞄准癌转移目标 诸平 Figure 1. Illustration of (a) the dual-ligand nanoparticle and (b) targeting of the nanoparticles to metastatic sites usingvascular targeting and a dual-ligand strategy. Inset: Interactions of circulating tumor cells and vascular bed. Figure 2. Evaluation of the ability of the dual-ligand nanoparticles to target metastasis in vivo in the MDA-MB-231 mousemodel. (a) The synopsis shows the timeline and schedule of the in vivo imaging studies. After 25 days from systemic injectionof MDA-MB-231 cells via the tail vein, bioluminescence imaging (BLI) showed the development of metastasis in the lungs.Each metastatic site was numbered, which is indicated on the BLI images. (b) Representative fluorescence moleculartomography (FMT) images of the mouse with metastatic spots 4 and 5. FMT imaging was performed 3 h after injection of acocktail of RGD-NP, PSN-NP, and dual-ligand-NP. (c) Using the different NIR fluorophores on each nanoparticle variant, thefluorescence signal in the thoracic region of the FMT images was quantified for each formulation (n = 5 mice). On the basis ofphantom measurements of each formulation using the FMT system, the fluorescence signal was converted to nanoparticleconcentration (mean ( SD; y-axis is in logarithmic scale). (d) The total number of nanoparticles for PSN-NP, RGD-NP, and dualligand-NPis shown for each metastatic spot (y-axis is in logarithmic scale). Figure 3. Evaluation of the ability of the dual-ligand nanoparticles to target metastasis in vivo in the 4T1 mouse model. (a) Thedifferent protocols of in vivo imaging and ex vivo and histological analysis are shown. (b) BLI images show a mouse before andafter resection of the primary tumor. (c) ROIs indicate the location of the different organs in an FMT image. (d) RepresentativeFMT images of a mouse with 4T1 metastasis are shown 3 h after injection of a cocktail of RGD-NP, PSN-NP, and dual-ligand-NP.After thresholding, the fluorescence signal of each formulation was color-coded (green: RGD-NP; red: PSN-NP; blue: dualligand-NP).(e) Using a CRi Maestro fluorescence imaging system, ex vivo imaging of lungs indicated the colocalization of thetargeted nanoparticles and 4T1 metastatic cells expressing GFP. (f) Using the ex vivo images to confirm the location ofmetastatic sites in each mouse, the targeting accuracy of the RGD-NP, PSN-NP, and dual-ligand-NP was calculated as apercentage of the metastatic sites being successfully captured by each formulation (n = 7 mice; *p 0.05 by Student's t test) Figure 4. Spatiotemporal targeting of P-selectin and Rvβ3 integrin in vivo in the 4T1 mouse model. (a) Representative FMTimages show targeting of nanoparticles to micrometastases 1 h postinjection in a mouse bearing 4T1 breast cancermetastasis. Mice with 4T1 metastasis (n = 8 mice) were injected with a cocktail of Rvβ3 integrin-targeting nanoparticles (RGDNP),P-selectin-targeting nanoparticles (PSN-NP), and nontargeted nanoparticles (NT-NP) containing an equal number ofparticles per formulation. (b) Quantification of the fluorescence signal from hot spots in the FMT images is shown for eachformulation. Each animal presented 24 hot spots (total 22 hot spots). The animals were imaged at three different time points(t = 19, 22, and 26 days after tumor inoculation) to capture an early stage and later stages of metastatic disease. (c) Two hotspots from the same animal show the time-course of signal for RGD-NP and PSN-NP. (d) Comparison of the signal for RGD-NPand PSN-NP was performed for each hot spot in the early-stage (t = 19 days) and late-stage disease (t = 26 days). To consider asignal of a targeted nanoparticle superior than that of the other formulation, it had to exhibit a difference of 100 pmol of thefluorophore or greater, which corresponded to the considerable difference of 4 1011 nanoparticles (～10% of the injecteddose). Only signals above 80 pmol of fluorescence were included in the analysis, since this was the detection threshold for the“nonspecific” signal of the nontargeted formulation. Figure 5. Targeting early stage metastasis using the dual-ligand nanoparticle. (a) The timeline and schedule is shown for thein vivo imaging studies and post mortem analyses. (b) Using whole-body planar gamma scintigraphy, a representative coronalimage shows the accurate targeting of a 99mTc-labeled dual-ligand-NP to early-stage metastases in the lungs of a mouse with4T1 metastasis 2 h after injection. Following a systemic injection of ～20 μCi of 99mTc-dual-ligand-NP, the animals were imagedusing a Gamma Medica X-SPECT system. The animal model was used 9 days after orthotopic tumor inoculation in a mammaryfat pad, which was the time point of early onset of lung metastasis. (c) In the end of the in vivo imaging session, the lungs of theanimals were perfused, excised, and imaged ex vivo using planar gamma scintigraphy, planar fluorescence imaging, and 3Dcryoimaging. 3D-cryoimaging provided an ultra-high-resolution fluorescence volume of the lungs showing the early onsetand topology of metastatic disease. (d) Overlaying ex vivo planar fluorescence and scintigraphy imaging of lungs, thecolocalization of dual-ligand-NP and 4T1 metastatic cells is shown. (e) The targeting efficacy of dual-ligand-NP was comparedin mice bearing 4T1 metastasis and healthy mice (n = 4). Using gamma scintigraphy, both groups of animals were imaged 2 hafter systemic injection of ～20 μCi of 99mTc-dual-ligand-NP. The signal intensity in the thoracic region was comparedquantitatively between the two groups. 据美国化学会主办的《化学与工程新闻》（ CEN ）周刊网站 2015 年 8 月 26 日 报道，美国凯斯西储大学（ Case Western Reserve University ）生物医学工程系、放射学系（ Department of Radiology ）、成像研究案例中心（ Case Center for Imaging Research ）以及综合性肿瘤中心（ Comprehensive Cancer Center ）的研究人员合作 , 采用双配位纳米粒子（ dual-ligand nanoparticle ）将两种在癌细胞内发现的转移性标记作为目标，追踪肿瘤细胞其从原发位向其他位的转移。图 1 就是肿瘤目标。对小鼠乳腺组织接种乳腺癌细胞之后 9 天内，此鼠的肿瘤发展转移到肺部。放射性标记的纳米粒子将2种早期转移突出明亮的恶性细胞标记作为目标。 Fig. 1 TARGETING TUMORS Metastatic tumors developed in the lungs of this mouse within nine days after its mammary tissue was seeded with breast cancer cells. Radioactively labeled nanoparticles that target two markers of early-stage metastasis highlighted the malignant cells. Credit: ACS Nano 尽管大多数癌症疗法治疗肿瘤是将其作为一个整体来进行处理 , 但是，细胞进化和行为改变是随时间而变化的。例如 , 它们可以改变其基因表达模式，以逃离原发肿瘤和扩散到全身。现在 , 研究者们已经开发出一种纳米微粒 , 在转移的2个不同阶段将肿瘤细胞作为目标 , 这有可能可以防止肿瘤扩散，相关研究结果于 2015 年 8 月 23 日 已经在《美国化学会纳米》（ ACS Nano ）杂志网站发表——详见 Elizabeth Doolittle , Pubudu M. Peiris , Gilad Doron , Amy Goldberg , Samantha Tucci , Swetha Rao , Shruti Shah , Meilyn Sylvestre , Priya Govender , Oguz Turan , Zhenghong Lee , William P. Schiemann , Efstathios Karathanasis . Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis . ACS Nano , 2015, 9(8): 8012-8021. DOI: 10.1021/acsnano.5b01552 . Publication Date (Web): July 23, 2015. 良性肿瘤无转移。恶性肿瘤则很容易发生转移，其转移方式有 4 种 : ①直接蔓延到邻近部位 ; ②淋巴转移 : 原发癌的细胞随 淋巴引流 ，由近及远转移到各级淋巴结，也可能超级转移 ; 或因癌阻碍顺行的淋巴引流而发生逆向转移。 转移癌 在 淋巴结 发展时， 淋巴结肿大 且变硬，起初尚可活动，癌侵越包膜后趋向固定，转移癌阻碍局部组织淋巴引流，可能引起皮肤、皮下或肢体的淋巴水肿 ; ③血行转移 : 癌细胞进入血管随血流转移至远隔部位如肺、肝、骨、脑等处，形成 继发性肿瘤 ; ④种植 : 瘤细胞脱落后种植到另一部位，如内脏的癌播种到腹膜或胸膜上。显然，恶性肿瘤转移将增加对机体的损害作用，而且影响转归。 癌症死亡人数当中大约有 90% 的人并不是由于最初的肿瘤而导致死亡，而是因为次生肿瘤即肿瘤转移所致 , 经常在肺部、骨骼、肝脏以及大脑中扎根。凯斯西储大学生物医学工程师埃夫斯塔西奥斯·卡拉塞纳希斯（ Efstathios Karathanasis ）指出，这些转移性细胞一般会幸免于化疗，“在体内隐藏于大量的健康细胞之中，要彻底灭杀干净，其药物浓度会如此之高 , 以至于会置病人于死地。 ”如何实现在有效灭杀癌细胞的同时，又不会伤及健康细胞，这一直是科学家研究重点。 通过在纳米微粒内包装小分子药物 , 有很多研究人员都希望开发出，仅仅为肿瘤细胞提供高浓度治疗剂量药物 , 同时有不会对健康组织造成影响的方法。埃夫斯塔西奥斯·卡拉塞纳希斯等人，他们通过一种配体来修饰承载药物的纳米微粒，将在癌细胞上发现的一种标识物作为靶标。埃夫斯塔西奥斯·卡拉塞纳希斯想进一步完善这种活动目标的方法。他说，癌症生物学建议针对一种癌症标志物是不够的。因为，来自同一个病人、同一肿瘤的所有癌细胞 , 在给定某一时间内，并非单一的特征蛋白质受体会出现过表达。所以，他决定以2种蛋白质标记物作为目标，这 2 种蛋白质标记物是肿瘤细胞经过了早期阶段的转移，具有不同阶段肿瘤细胞的特征。 为了验证这一想法 , 他的研究团队用配体修饰了直径为 100 nm 的脂质体 , 其目标是逃离肿瘤之后的转移性癌细胞2种表面蛋白表达。此逃离肿瘤之后的转移性癌细胞，进入血管随血流循环，再转移至远隔肿瘤原位的其他部位如肺、肝、骨、脑等处，形成 继发性肿瘤 ，这种肿瘤细胞的转移是属于血行转移。此 2 种蛋白质有助于血液循环肿瘤细胞退出血流，在一个新位点安营扎寨，这样它们就可以建立一个新肿瘤即形成 继发性肿瘤 。有一种被称之为选择素（ selectin ）的蛋白质 , 帮助肿瘤细胞在血液中循环，开始沿着血管的内壁向前滚动。而第二种蛋白质是整合素（ integrin ） , 它帮助这些肿瘤细胞在退出血流和滋生新的肿瘤之前，牢牢地附着在血管上。 选择素（ selectin ）是一类异亲型结合、 Ca 2+ 依赖的 细胞粘着分子 ，是 细胞黏附分子 中的一个家族，为 I 型单链跨膜 糖蛋白 ，能与特异糖基识别并结合。主要参与 白细胞 与 血管内皮细胞 之间的识别与粘着。选择素主要有血小板选择素（ platelet selectin ）、内皮细胞选择素（ endothelial selectin ）、白血球选择素（ leukocyte selectin ）。选择素又被称为 选择蛋白 或选择 凝集素 。 整合素（ Integrin ）又被称为 整联蛋白 ，是一种介导细胞和其外环境（如 细胞外基质 ， extracellular matrixc 简称 ECM ）之间连接的跨膜受体。在信号 转导 中，整合素将 ECM 的化学成分与力学状态等有关信息传入细胞。因此，整合素除了穿过膜的机械作用，也参与了细胞讯息、 细胞周期 的调节、细胞型态以及细胞的运动。通常，受体的作用是将外环境的变化通知细胞并引起细胞反应。但整合素不仅介导由外到内的信号，也介导由内到外的细胞信号。因此整合素不但将 ECM 的信息传递给细胞，也将细胞的状态表达给外界，从而可以迅速和灵活地响应环境中的变化，比如血液的凝固作用等。细胞外基质是由细胞分泌到细胞外间质中的大分子物质，构成复杂的网架结构，支持并连接组织结构、调节组织的发生和细胞的生理活动。 为了能够验证纳米颗粒能否找到恶性癌细胞 , 埃夫斯塔西奥斯·卡拉塞纳希斯研究小组对其在2个不同的转移三阴性乳腺癌小鼠模型身上进行了测试。他们对实验鼠注射了荧光性或放射性标记的纳米微粒药剂 , 发现此纳米微粒追踪并击中了标记物目标。卡拉塞纳希斯说，他们发现大约有 90% 的微转移点，即大小在 10 ～ 30 μm 的癌细胞簇已被捕获。因为整合素和选择素也是炎症和心血管疾病的标志物 , 研究人员将需要测试的此纳米微粒的副作用。除此之外，他们还计划测试承载抗癌药物纳米微粒的识别系统。 乔治亚理工学院和埃默里大学（ Georgia Tech and Emory University ）生物医学工程系主任 拉维 ·贝拉姆康德 （ Ravi V. Bellamkonda ）对双重目标的策略印象深刻。他认为， “ 应该认识到肿瘤并非铁板一块 , 在每个肿瘤内或在每一个病人体内 , 可能都会有处于不同发展阶段或转移性扩散的肿瘤。 ” 将纳米治疗设计更好的与当前对于癌症生物学的理解归并在一起可能效果更佳。更多信息请浏览原文。 原文摘要如下： Abstract Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and α v β 3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.
Written by Sarah Waxma New York City’s Chinatown, the largest Chinatown in the United States—and the site of the largest concentration of Chinese in the western hemisphere—is located on the lower east side of Manhattan. Its two square miles are loosely bounded by Kenmore and Delancey streets on the north, East and Worth streets on the south, Allen street on the east, and Broadway on the west. With a population estimated between 70,000 and 150,000, Chinatown is the favored destination point for Chinese immigrants, though in recent years the neighborhood has also become home to Dominicans, Puerto Ricans, Burmese, Vietnamese, and Filipinos among others. Chinatown is born Chinese traders and sailors began trickling into the United States in the mid eighteenth century; while this population was largely transient, small numbers stayed in New York and married. Beginning in the mid nineteenth century, Chinese arrived in significant numbers, lured to the Pacific coast of the United States by the stories of “Gold Mountain” — California — during the gold rush of the 1840s and 1850s and brought by labor brokers to build the Central Pacific Railroad. Most arrived expecting to spend a few years working, thus earning enough money to return to China, build a house and marry. As the gold mines began yielding less and the railroad neared completion, the broad availability of cheap and willing Chinese labor in such industries as cigar-rolling and textiles became a source of tension for white laborers, who thought that the Chinese were coming to take their jobs and threaten their livelihoods. Mob violence and rampant discrimination in the west drove the Chinese east into larger cities, where job opportunities were more open and they could more easily blend into the already diverse population. By 1880, the burgeoning enclave in the Five Points slums on the south east side of New York was home to between 200 and 1,100 Chinese. A few members of a group of Chinese illegally smuggled into New Jersey in the late 1870s to work in a hand laundry soon made the move to New York, sparking an explosion of Chinese hand laundries. Living arrangements From the start, Chinese immigrants tended to clump together as a result of both racial discrimination, which dictated safety in numbers, and self-segregation. Unlike many ethnic ghettos of immigrants, Chinatown was largely self-supporting, with an internal structure of governing associations and businesses which supplied jobs, economic aid, social service, and protection. Rather than disintegrating as immigrants assimilated and moved out and up, Chinatown continued to grow through the end of the nineteenth century, providing contacts and living arrangements — usually 5-15 people in a two room apartment subdivided into segments — for the recent immigrants who continued to trickle in despite the enactment of the Chinese Exclusion Act of 1882. Immigration and Chinatown The Chinese Exclusion Act (1882-1943), to date the only non-wartime federal law which excluded a people based on nationality, was a reaction to rising anti-Chinese sentiment. This resentment was largely a result of the willingness of the Chinese to work for far less money under far worse conditions than the white laborers and the unwillingness to "assimilate properly". The law forbids naturalization by any Chinese already in the United States; bars the immigration of any Chinese not given a special work permit deeming him merchant, student, or diplomat; and, most horribly, prohibits the immigration of the wives and children of Chinese laborers living in the United States. The Exclusion Act grew more and more restrictive over the following decades, and was finally lifted during World War II, only when such a racist law against a wartime ally became an untenable option. “The Bachelor’s Society” The already imbalanced male-female ratio in Chinatown was radically worsened by the Exclusion Act and in 1900 there were only 40-150 women for the upwards of 7,000 Chinese living in Manhattan. This altered and unnatural social landscape in Chinatown led to its role as the “Bachelor’s Society" with rumors of opium dens, prostitution and slave girls deepening the white antagonism toward the Chinese. In keeping with Chinese tradition — and in the face of sanctioned U.S. government and individual hostility — the Chinese of Chinatown formed their own associations and societies to protect their own interests. An underground economy allowed undocumented laborers to work illegally without leaving the few blocks they called home. An internal political structure comprised of the Chinese Consolidated Benevolent Association and various tongs, or fraternal organizations, managed the opening of businesses, made funeral arrangements, and mediated disputes, among other responsibilities. The CCBA, an umbrella organization which drafted its own constitution, imposed taxes on all New York Chinese, and ruled Chinatown throughout the early and mid twentieth century, represented the elite of Chinatown; the tongs formed protective and social associations for the less wealthy. The On Leong and Hip Sing tongs warred periodically through the early 1900s, waging bloody battles that left both tourists and residents afraid to walk the streets of Chinatown. Growth in Chinatown When the Exclusion Act was finally lifted in 1943, China was given a small immigration quota, and the community continued to grow, expanding slowly throughout the ‘40s and ‘50s. The garment industry, the hand-laundry business, and restaurants continued to employ Chinese internally, paying less than minimum wage under the table to thousands. Despite the view of the Chinese as members of a “model minority,” Chinatown’s Chinese came largely from the mainland, and were viewed as the “downtown Chinese," as opposed the Taiwan-educated “uptown Chinese,” members of the Chinese elite. When the quota was raised in 1968, Chinese flooded into the country from the mainland, and Chinatown’s population exploded, expanding into Little Italy, often buying buildings with cash and turning them into garment factories or office buildings. Although many of the buildings in Chinatown are tenements from the late nineteenth and early twentieth centuries, the rents in Chinatown are some of the highest in the city, competing with the Upper West Side and midtown. Foreign investment from Hong Kong has poured capital into Chinatown, and the little space there is a precious commodity. Chinatown Today Today’s Chinatown is a tightly-packed yet sprawling neighborhood which continues to grow rapidly despite the satellite Chinese communities flourishing in Queens. Both a tourist attraction and the home of the majority of Chinese New Yorkers, Chinatown offers visitor and resident alike hundreds of restaurants, booming fruit and fish markets and shops of knickknacks and sweets on torturously winding and overcrowded streets. 原文见 http://www.chinatown-online.com/nychinatown/aboutchinatown.shtml
昨天是复活节假期的最后一天，貌似闲极无聊的我观看了同学进行western实验，虽说不完整，但本着好记性不如烂笔头的原则，要做个简单记录。 好吧那么什么是分子生物学者们经常挂在嘴边的western实验呢？抄一下维基的定义： The western blot (alternatively, protein immunoblot ) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF ), where they are probed (detected) using antibodies specific to the target protein. 翻译：western免疫印迹法是用于从样品组织匀浆或提取物中检测特定蛋白的一种分析技术。它使用凝胶电泳技术，基于多肽的长度（变性条件下）或者蛋白的三维结构（天然/未变性条件下）来分离天然的或者变性的蛋白。蛋白被电泳分离后被转移到一张一般由硝化纤维或聚偏氟乙烯制作的膜上，被对目标蛋白有特异性的抗体所探测。 下面是我对同学他们实验过程的记录。 动物组织（大鼠的肺脏）分别包在锡纸里，放在装满干冰的泡沫盒里，从德国寄来。接着，同学将样品取出，投入装着液氮的保温罐，低温冷冻。液氮温度是-140摄氏度。取出大理石研钵，将锡纸包好的样品用长镊子从液氮里夹出，用镊子另一端捣两下使之断裂，打开锡纸包装，将一块组织样本倒入事先剪掉一个小角的小自封塑料袋里，封口，立刻包上锡纸，放入研钵，倒入适量液氮，用石杵用力挤碎。注意不要研磨，只自上而下用力挤即可，以免把袋子和锡纸弄破，污染样品。感觉组织都彻底被压成粉末后，立刻取出，从那个剪下的小角处把组织粉末倒入事先写好标号的两个塑料小离心管里（一个用于提蛋白，一个用于提RNA），盖紧，立刻投入液氮中。这样研磨并分装好所有的组织样品。 接着，用镊子取出液氮里的一个小离心管，迅速加入300微升 Western细胞裂解液 ，放入普通冰中。涡旋十秒左右使之充分混合。如嫌不够，可以多涡旋几次。等待十五分钟左右。4摄氏度12000转（好像是）离心5分钟，取出上清液移入另外的新离心管里。上清液里蛋白浓度很高所以会比较粘稠，不好取，需要比较小心，必要时可以多次离心。这个过程可以不用冰，因为 裂解液里有某种成分，阻止蛋白继续降解 。 取出100微升上清液，用水稀释到500微升。之后，从这个稀释5倍的蛋白溶液中再取3微升到新离心管中，稀释到60微升，用于测定蛋白含量。每个样品取两个平行，也就是一共6微升。将20微升稀释液加入测蛋白含量用的盘子里（之前，盘中应该已经加过做线性用的不同浓度BSA标准品，也是两个平行）。最后将显色试剂（考马斯亮蓝？）用排枪小心加入盘中。避光放置10min,用紫外分光光度计在 595或550nm 下测定吸光度，电脑程序在你已经输入线性每一个浓度的情况下自动算线性，并且直接算出样品浓度。或者手动计算亦可。 我只观看到上面这里。下面的内容，据推测应该是：通过各样品的蛋白含量算出一个使各自蛋白含量相同的稀释过5倍的蛋白裂解液体积，大约使之为60微升左右；加入loading buffer(是干什么用的？）将此混合液体小心加入事先制备好的聚丙烯酰胺凝胶电泳的梳齿里，进行电泳；电泳结束后进行 转膜封闭杂交显色 等。其实western的基本操作，网上有详细的步骤： http://www.docin.com/p-2506990.html?RisingToken=16663116859320#docTitle 。但是具体看人做肯定和只看步骤不同。下次有机会再观察他们的下一步实验。 我问实验者：这个过程中什么对结果影响最大？做实验的姐姐回答： 速度 。原来前面提取过程中，处理样品要迅速，不能让它解冻，一解冻，蛋白就会立刻发生降解，结果就不准确了。
Western Blot Protocol SDS-PAGE 1. Clean and dry the glass plates and comb. 【 Optional: 1.0mm, 1.5mm and 10-well, 15-well 】 2. Assemble the gel-casting unit. ? Form the gel sandwich by assembling the spacers and two glass plates in the clamps. ? Align the bottom part of the spacers and two glass plates at the same level, and then tighten the clamp. ? Place the gel sandwich onto the casting stand. ? Insert the comb, and mark the glass plate at a level ~1.0-1.5 cm below the bottom of the comb teeth. 3. Pour the resolving gel. ? Prepare the appropriate resolving gel mixture using the recipes in Table 1. Make sure that the solution is well mixed before adding the TEMED. ? Use a 10-ml pipette to transfer the mixture to the glass-plate sandwich up to the marked level. ? Carefully overlay the gel with a ~2-mm-deep layer of 75% ethanol. 【 This prevents air from reaching the gel, which inhibits polymerization of the acrylamide, and ensures that the gel surface is flat. 】 ? After polymerization is complete (~30 min), pour off the overlaying ethanol, wash the gel surface with H 2 O, and carefully remove any remaining liquid with filter paper without damaging the gel surface. 【 Polymerization of the gel is evidenced by a clear refractive index change that can be seen between the gel and the overlay liquid, or refer to the remainder of the prepared gel solution. 】 4. Pour the stacking gel. ? Select an acrylamide concentration for the stacking gel, and make the appropriate mixture, using the recipes in Table 2. Make sure that the solution is well mixed. ? Carefully overlay the resolving gel with the stacking gel solution until the height of the stacking gel is ~2.0-3.0 cm . ? Insert the comb into this solution, leaving 1.0-1.5 cm between the top of the resolving gel and the bottom of the comb. Make sure that no air bubbles are trapped beneath the teeth of the comb. ? Allow the stacking gel mixture to polymerize for ~30 min. 5. Assemble the cassette in the electrophoresis apparatus according to the manufacturers instructions. Fill the apparatus with 1 Running B uffer ensuring that the buffer fully fills the sample loading wells and the bottom of the gel. Carefully remove the sample comb from the stacking gel. 6. Preparation of Samples. ? Mix the protein solution with SDS Loading Buffer (5) in a 4:1 ratio. 【 Add 50l - mercaptoethanol to 1ml SDS Loading Buffer before use. 】 ? Heat the samples in a heat block for 2 minutes at 95C to denature the proteins and ensure the maximum amount of SDS binding to the proteins. Allow the samples to cool to room temperature. Remove insoluble materials by centrifugation. 7. Running a Discontinuous Slab Gel. ? Use a pipette to load the samples into the sample well. ? Connect the power supply to the electrophoresis apparatus with the anode (+) linked with the bottom reservoir and the cathode (-) connected to the upper reservoir. ? Pass a constant voltage of 50-130 V at room temperature , through the gel until the Bromophenol Blue dye front reaches the bottom of the gel. ? Turn off the power supply, and disconnect the electrodes. Remove the gel plates from the apparatus, and carefully remove a spacer. Use the spacer to gently pry the gel plates apart, leaving the gel stuck to one plate. Immunoblot 8. Electro-transfer to nitrocellulose （ NC 膜） or 硝酸纤维素（ PVDF ）。 ? Carefully transfer the gel to clamp in p re-chilled transfer buffer in a bottom-to-top order of filter paper, gel, nitrocellulose membrane, filter paper, sponge. ? Start transferring by adding transfer buffer and adjust ing to 250mA (constant current) o r 100-120V and set the timer 60 -120 min. 9. After transfer, cut the nitrocellulose membrane into sub-pieces according to the pre-stained protein markers. 【 Mark the membranes in order to distinguish them. 】 10. Block the membrane with 5% milk-TBST for 60min or longer . 11. Incubate the membranes in desired primary antibodies at room temperature for 60min or longer if cold , wash with 1 TBST buffer 3 times (15min/time). 12. Incubate the membranes in appropriate HRP-conjugated second antibodies at room temperature for 60min or longer if cold , wash with 1 TBST buffer 3 times (15min/time). 13. Prepare an appropriate volume of HRP substrate solution, add onto the membranes, and allow reaction for ~30s. 14. Cast membranes in a clean X-ray film box for image. Western Blotting Reagents IN AN ORDER OF USE Loading buffer glycerol Sodium dodecyl sulfate (SDS), Sigma L4390 -mercaptoethanol, Wolsen bromophenol blue Gel preparation ddH 2 O Acrylamide, Wolsen Bis-Acrylamide, Wolsen Tris, ShangHai RiChu BioScience Co.,Ltd HCl Sodium dodecyl sulfate (SDS), Sigma L4390 Ammonium persulfate, Sigma A9164 N, N, N, N,-Tetramethylethylenediamine, Sigma T7024 Ethanol absolute, TianJing Fuyu Electrophoresis Tris, ShangHai RiChu BioScience Co.,Ltd Glycine, Wolsen Sodium dodecyl sulfate (SDS), Sigma L4390 Page Ruler TM Prestained Protein Ladder Transfer Tris, ShangHai RiChu BioScience Co.,Ltd Glycine, Wolsen Ethanol absolute, TianJing Fuyu Nitrocellulose membrane Filter paper Block, immunoblot and wash Nonfat milk, 伊利 Tris, ShangHai RiChu BioScience Co.,Ltd Sodium chloride (NaCl), Sinopharm Chemical Reagent Co., Ltd Tween-20, BETTER 0777 Buffer Preparation ? 30%T (2.6%C) Acrylamide stock solution 29.22 g acrylamide 0.78 g bisacrylamide 100 ml H 2 O Filter the stock solution through Whatman filter paper and store at 4C. ? Tris-HCl 1.5M (pH 8.8)500ml: 90.86 g Tris, adjust to pH 8.8 with HCl. 1.0M (pH 6.8)100ml: 12.11 g Tris, adjust to pH 6.8 with HCl. ? 10% SDS 1L 100g SDS Heat to 68C for solubility. pH ~6.6 ? Ammonium persulfate solution (10%, w/v) 1 g ammonium persulfate in 10 mL H 2 O and store at 4C. ? Tris-glycine Running Buffer (10)1L 10: 250 mM Tris, 1.92 M glycine, 1% SDS 1: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 30.3 g Tris 144 g glycine 10 g SDS No need to adjust pH. ? Tris-HEPES-SDS Running Buffer (10)1L 1: 100 mM Tris, 100 mM HEPES, 3 mM SDS 121 g Tris 238 g HEPES 10 g SDS ? SDS Loading Sample Buffer (5)100 ml 250 mM Tris-HCl (pH6.8), 10% SDS, 30% Glycerol, 5% -mercapitalethanol (or 0.5M DTT), 0.5% bromophenol blue ? Western transfer buffer (1)1L 1: 25 mM Tris, 192 mM glycine, pH8.3 3.03 g Tris 14.4 g glycine 200 ml absolute ethyl alcohol No need to adjust pH. Dissolved in ~500 ml ddH 2 O, then add absolute ethyl alcohol. ? Western blotting block buffer 5% Nonfat milk in TBS - T 5g milk in 100 ml TBST ? Tris buffered saline-Tween (TBST) wash buffer (10)1L High salt 24.2 g Tris 80 g NaCl 10 ml Tween-20 HCl to adjust pH 7.6-8.0 Low salt 24.2 g Tris 8 g NaCl 10 ml Tween-20 HCl to adjust pH 7.6-8.0 Table 1 Preparing resolving gels for Tris-glycine SDS-polyacrylamide gel electrophoresis （分离胶） Gel Volume 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml 6% gel H2O 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5 30% acrylamide mix 1 2 3 4 5 6 8 10 Tris-Cl (1.5 M, pH 8.8) 1.3 2.5 3.8 5 6.3 7.5 10 12.5 SDS (10%) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04 8% gel H2O 2.3 4.6 6.9 9.3 11.5 13.9 18.5 23.2 30% acrylamide mix 1.3 2.7 4 5.3 6.7 8 10.7 13.3 Tris-Cl (1.5 M, pH 8.8) 1.3 2.5 3.8 5 6.3 7.5 10 12.5 SDS (10%) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED 0.003 0.006 0.009 0.012 0.015 0.018 0.024 0.03 10% gel H2O 1.9 4 5.9 7.9 9.9 11.9 15.9 19.8 30% acrylamide mix 1.7 3.3 5 6.7 8.3 10 13.3 16.7 Tris-Cl (1.5 M, pH 8.8) 1.3 2.5 3.8 5 6.3 7.5 10 12.5 SDS (10%) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 12% gel H2O 1.6 3.3 4.9 6.6 8.2 9.9 13.2 16.5 30% acrylamide mix 2 4 6 8 10 12 16 20 Tris-Cl (1.5 M, pH 8.8) 1.3 2.5 3.8 5 6.3 7.5 10 12.5 SDS (10%) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 15% gel H2O 1.1 2.3 3.4 4.6 5.7 6.9 9.2 11.5 30% acrylamide mix 2.5 5 7.5 10 12.5 15 20 25 Tris-Cl (1.5 M, pH 8.8) 1.3 2.5 3.8 5 6.3 7.5 10 12.5 SDS (10%) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 10% ammonium persulfate 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 Table 2 Preparing 5% stacking gels for Tris-glycine SDS-polyacrylamide gel electrophoresis Gel Volume 1 ml 2 ml 3 ml 4 ml 5 ml 6 ml 8 ml 10 ml H2O 0.68 1.4 2.1 2.7 3.4 4.1 5.5 6.8 30% acrylamide mix 0.17 0.33 0.5 0.67 0.83 1 1.3 1.7 Tris-Cl (1.0 M, pH 6.8) 0.13 0.25 0.38 0.5 0.63 0.75 1 1.25 SDS (10%) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 ammonium persulfate (10%) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 TEMED 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01
Tris 分子式:(HOCH 2 ) 3 CNH 2 分子量.:121.14 (1)规格: 外观：白色结晶 含量：99.5% 熔点：168-172℃ 水不溶物：0.01% 强热残份：0.05% 重金属(aspb)：5PPM 干燥失重：0.2% 吸光度(260nm)：0.2 PH(0.1mol/L,25℃)：10.0-10.8 Fe：1PPM SO 4 2+ ：5PPM Cl：5PPM Appearance:whitecrystal Content:99.5% Meltingpoint:168-172℃ Lossondrying:0.2% ASH:0.05% PH(0.1mol/L,25℃):10.0-10.8 InsolubleinWater:0.01% ABS(260nm):0.2 Heavymetals:5PPM Fe:1PPM SO 4 2+ ：5PPM Cl:5PPM 别名:三羟甲基氨基甲烷;2-氨基-2-羟基-1,3-丙二醇;氨丁三醇;缓血酸铵;三甲醇氨基甲烷;Trishydroxymethylaminomethane;2-amino-2-hydroxymethylpropanediol;THAM;TRIS;trisbase;Trisbuffer;freebase;Tris(hydroxymethyl)methylamine;Trizma;trizmabase;Trometamol;Tromethamine;Tromethane Usage:用作缓冲剂。Tromethamineisusedasanintermediateforthepreparationofsurfaceactiveagents,vulcanizationaccelerators,andpharmaceuticals,andusedasatitrimetricstandard. 不可以高压灭菌！绝对不行！ 缓冲液最重要的是pH值，加NaCl并不影响pH，按照配方加入就行了。 Acrylamide ： A colorless, odorless, highly water soluble vinyl monomer formed from the hydration of acrylonitrile. It is primarily used in research laboratories for electrophoresis, chromatography, and electron microscopy and in the sewage and wastewater treatment industries. Bis-Acrylamide 甲叉双丙烯酰胺（或双丙烯酰胺） TEMED（ tetramethylethylenediamine ） n. 【有机化学】四甲基乙二胺 SDS （Sodium Dodecyl Sulfate）是阴离子去污剂，作为变性剂和助溶试剂，它能断裂分子内和分子间的氢键，使分子去折叠，破坏蛋白分子的二、三级结构。 ammonium persulfate 过硫酸铵
JGR Eitors Highlight Trend discrepancies among three best track data sets of western North Pacific tropical cyclones Jin-Jie Song School of Atmospheric Sciences and Key Laboratory of Mesoscale Severe Weather/Ministry of Education, Nanjing University, Nanjing, Jiangsu, China Yuan Wang School of Atmospheric Sciences and Key Laboratory of Mesoscale Severe Weather/Ministry of Education, Nanjing University, Nanjing, Jiangsu, China Liguang Wu Key Laboratory of Meteorological Disaster of the Ministry of Education, Nanjing University of Information Science and Technology, Nanjing, Jiangsu, China The hot debate over the influence of global warming on tropical cyclone (TC) activity in the western North Pacific over the past several decades is partly due to the diversity of TC data sets used in recent publications. This study investigates differences of track, intensity, frequency, and the associated long-term trends for those TCs that were simultaneously recorded by the best track data sets of the Joint Typhoon Warning Center (JTWC), the Regional Specialized Meteorological Center (RSMC) Tokyo, and the Shanghai Typhoon Institute (STI). Though the differences in TC tracks among these data sets are negligibly small, the JTWC data set tends to classify TCs of category 23 as category 45, leading to an upward trend in the annual frequency of category 45 TCs and the annual accumulated power dissipation index, as reported by Webster et al. (2005) and Emanuel (2005). This trend and potential destructiveness over the period 19772007 are found only with the JTWC data set, but downward trends are apparent in the RSMC and STI data sets. It is concluded that the different algorithms used in determining TC intensity may cause the trend discrepancies of TC activity in the western North Pacific. Received 22 August 2009; accepted 29 January 2010; published 30 June 2010. Citation: Song, J.-J., Y. Wang, and L. Wu (2010), Trend discrepancies among three best track data sets of western North Pacific tropical cyclones, J. Geophys. Res., 115, D12128, doi:10.1029/2009JD013058. Check out Figure 1 of the paper at least, if you are interested in the trend. Click here for the pdf file of the paper
标签: western
