电转染标准步骤 (仅供参考)-临床医学研究中心

实验方法Experimental Methods

1. 清洗电转杯,将电转杯置于75%酒精中浸泡2小时,然后在紫外灯下照射后即可使用。

2. 电穿孔前一天,以合适密度传代细胞,使细胞在转染前出于对数生长期。对于原代培养细胞,一般培养2-3天后,细胞单层融合度可以达到70-80%,即可开始试验。

3. PBS洗细胞2次,胰酶消化细胞2min,细胞变圆后,用10%血清培养基终止消化。

4. 轻轻吹下细胞成单细胞悬液后,1200rpm室温离心5min。

5. 弃上清,加无血清培养基重悬离心。根据细胞数量,加入适量的无血清培养基重悬细胞,并上下吹打均匀。

注:此时进行细胞计数,使细胞量达到1-3*106cells/ml

6. 加入适量相应浓度的待转染核酸至相应的终浓度(质粒为20ug/ml,SiRNA的终浓度为100nM),上下吹打均匀。若以0.5ml计算,所需质粒为0.5×20=10ug。

7. 将混合液移入相应规格的电转杯中,按照预定条件设置参数,进行电击操作。(不同细胞电转前,需要进行预试验摸索电转的最佳条件,既要保证较高的转染效率,又要保证较高的细胞存活率。经过实践摸索后得出,相应的最佳参数为:质粒:250V,10ms,1(最多2),0,4mm cuvette;SiRNA: 250V,5ms,1(最多2),0,4mm cuvette。

8. 电击完成后,将电转杯置于恒温培养箱中8-12min,以使核酸充分进入细胞。

9. 将电击杯从恒温培养箱中取出,接种细胞悬液于室温10%培养基中,上下吹打均匀后,置于培养箱中正常培养。

10. 培养24h后,观察细胞存活状况。

11.48h后,即可进行细胞转染效率的检测或是进行细胞的实验处理。

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根折的治疗原则_腾讯新闻

根折的治疗原则

2021
05/13
12:00
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根折的治疗首先应是促进其自然愈合,即使牙齿似乎很稳固,也应尽早用夹板固定,以防活动。除非牙齿外伤后已数周,松动度较小可不必固定。

一般认为根折越靠近根尖其预后越好。当根折限于牙槽内时,对预后是很有利的,但折裂累及龈沟或发生龈下折时,常使治疗复杂而且预后亦差。

对根尖1/3折断,在许多情况下只上夹板固定,无需牙髓治疗,就可能出现修复并维持牙髓活力。但当牙髓有坏死时,则应迅速进行根管治疗术。

对根中1/3折断可用夹板固定;如牙齿冠端有错位时,在固定前应复位。复位固定后,每月应复查1次,检查夹板是否松脱,必要时可更换夹板。复查时若发现根折冠段牙髓坏死,应及时拔髓。如根折根尖段牙髓仍有活力,则只需作根折冠段的根管治疗术;若根折根尖段牙髓已坏死,就应一并作根管治疗术。

颈侧1/3折断并与龈沟相交通时,将不会出现自行修复。如牙根长度足以进行桩冠修复时,可用切龈术,或用正畸牵引法或牙槽内牙根移位术,将牙根断端牵出暴露于龈上以便修复。

纵行根折的预后不佳,往往需拔牙。有时可试行根管治疗术后,作牙体半切除术或截根术。

往期推荐

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前牙外伤应急处理病例

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A fixation method for the optimisation of western blotting | Scientific Reports

Results

Fixation Prior to Immunostaining

To optimise the fixation using organic solvent and heating, we varied the temperature and used different membranes (Fig. 1). Firstly, different fixation treatments on PVDF and nitrocellulose membranes were tested (Fig. 1a). Pooled human serum sample, containing 10 μg of protein, was resolved using 10% SDS-PAGE. Following this, the samples were used for Coomassie Brilliant Blue (CBB) staining (lane i) or electroblotted onto PVDF (Fig. 1a, top panel) or nitrocellulose membrane (Fig. 1a, bottom panel), then incubated with IgG antibody after the application of different fixative treatments, including no fixation (lane ii); drying at room temperature (lane iii); heating at 50 °C (lane iv); heating at 100 °C (lane v); immersion into the organic solvents (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) (lane vi) at room temperature; immersion into the organic solvents at 0 °C (lane vii); immersion into the organic solvents at 0 °C followed by sample heating at 100 °C (lane viii) and immersion into the organic solvents at 0 °C followed by sample heating at 50 °C (lane ix). The results obtained using the PVDF membranes showed that every treatment has an effect on preventing protein loss from electroblotted membranes and increase its retaining compared to the traditional method, the difference in improving detection sensitivity between various fixation treatments and traditional method were analysed and showed in the right graph. These treatments lead to a higher intensity of the IgG signal, especially for that of the IgG light chain, a relatively low molecular weight protein. Furthermore, heating at 50 °C for 30 min (lane iv) was shown to improve IgG detection more than drying at room temperature (20–25 °C) (lane iii) or heating at 100 °C for 30 min (lane v). Acetone treatment at 0 °C (lane vii) led to better results than acetone treatment at room temperature for 30 min (lane vi), while the optimal condition of all was acetone treatment at 0 °C followed by heating at 50 °C (lane ix), both for 30 min. Furthermore, a large amount of protein was preserved on the membrane after fixation and we were able to detect degradation products of human IgG subclasses17 (asterisk, Fig. 1a, lane ix). These fragments were confirmed by analysing at least 30 μg of serum protein without the fixation step. To obtain a similar signal intensity to that of samples that underwent fixative treatment, 50 μg of serum protein was required for the analysis without applying the fixation step (Supplementary Fig. 1). We performed similar experiments using nitrocellulose membranes (Fig. 1a, bottom panel). Due to the different characteristics of nitrocellulose and PVDF membranes, different fixation procedures are required. Since nitrocellulose membranes dissolve in methanol, acetone, and other organic solvents18, we tested a series of acetone or methanol solutions, using water as the solvent. Of all the investigated fixatives, the application of 75% methanol, 50% acetone, and sample heating at 50 °C improved protein fixation the most. However, these treatments resulted in high intensity background signals. Optimal fixation was achieved when the membranes were incubated with 50% methanol/water (v/v) solution at 0 °C, followed by heating at 50 °C, both for 30 min.

Figure 1

Fixation method-dependent differences in the immunostaining intensity. (a) Pooled human serum samples (10 μg) were analysed by 10% SDS-PAGE, and the separated proteins were transferred onto PVDF (top) and nitrocellulose (bottom) membranes, the whole membrane was cutted into eight pieces for subsequently fixation treatments. Representative images, showing anti-human IgG antibody staining after the following treatments: lane i, Coomassie Brilliant Blue (CBB) staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, heating at 50 °C; lane v, heating at 100 °C; lane vi, immersion into the organic solvents (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) at room temperature; lane vii, immersion into the organic solvents at 0 °C; lane viii, immersion into the organic solvents at 0 °C followed by sample heating at 100 °C; lane ix, immersion into the organic solvents at 0 °C followed by sample heating at 50 °C. All treatments were performed for 30 min. The relative intensity was shown on the right (n = 3 individual experiments). (b) Proteins were separated by 10% SDS-PAGE, and transferred onto PVDF membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments. Fixed in acetone at 0 °C, and heated at different temperatures for 30 min prior to the immunostaining. Lane i, CBB staining; lane ii, no fixation; lane iii, heating at 25 °C; lane iv, heating at 50 °C; lane v, heating at 75 °C; lane vi, heating at 100 °C. Right panel shows the relative intensity at different temperatures. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).

Additionally, we investigated the temperature-dependence of the PVDF fixation process (Fig. 1b), the treatments including no fixation group (lane ii); heating at 25 °C (lane iii); heating at 50 °C (lane iv); heating at 75 °C (lane v); heating at 100 °C (lane vi). Results show that retained proteins were increased in the all tested temperatures compared to the traditional method. Furthermore, results indicate that the signal intensity of IgG markedly increased at 50 °C compared to the intensity at 25 °C. However, the detection of the protein bands decreased gradually with the increase in temperature beyond 50 °C. This demonstrated that the optimal fixation temperature for detection using anti-IgG antibody is 50 °C.

Effects of the fixation step on sensitivity of immunoblotting

The sensitivity of immunoblotting coupled with the optimised fixation method was determined. Serial dilutions of the pooled human serum were separated by 10% SDS-PAGE, and the proteins were transferred onto PVDF membranes. These membranes were stained with anti-IgG, anti-haptoglobin (HP), anti-α2,6-sialyltransferases (ST6Gal1), and anti-eukaryotic elongation factor 1 alpha 2 (EEF1A2) antibodies, without pre-treatment (Fig. 2a, left) or following the optimised fixation method (Fig. 2a, right). The results showed that without the fixation step, 5 μg of serum protein was required for the visualisation of HP and IgG, two highly abundant proteins, while with the fixation step, the required amount was 1.3 μg of proteins, showing approximately four-fold increase in protein detection. The intensity of the methodology for the detection of ST6Gal1 and EFF1A2, proteins with low abundance in sera, was shown to increase two-fold following sample fixation. The intensity analysis of the retention of proteins following the fixation were shown to increase 1.8- to 16-fold, compared with those in the samples without pre-treatment (Fig. 2b). Similar results were obtained using nitrocellulose membranes (Supplementary Fig. 2), where the intensity increased approximately 1.6- to 7.6-fold.

Figure 2

Effects of sample fixation on the retention of proteins of the method using PVDF membranes. (a) Indicated numerals are amounts (5.0, 2.5, 1.3, and 0.6 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. The blotted membranes were treated using the traditional (left panel) or optimised fixation protocol (right). (b) Staining intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, optimised fixation protocol. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. *Significantly different p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).

Sample fixation prior to LB

Pooled human serum protein samples (3 μg) were used for the optimization of the fixation step coupled with the lectin blotting (LB) procedure with Lens culinaris agglutinin (LCA) and Sambucus nigra agglutinin (SNA) lectins (Fig. 3). All applied fixation methods (Fig. 3, lanes iii-vi) allowed an effective prevention of protein removal from the membranes during LB, and the difference in improving detection sensitivity between various fixation treatments and traditional method were analysed and showed in the right graph. The optimal fixation method for PVDF membranes, was acetone treatment at room temperature followed by sample heating at 100 °C, both for 30 min. In the case of nitrocellulose membranes, the optimal fixation method was a combination of incubation in 50% methanol solution and heating at 100 °C for 30 min each. More protein bands were observed while using PVDF membranes, in both cases of procedures with and without fixation, compared with those observed when using nitrocellulose membranes. This discrepancy is due to the higher mobility of low molecular-weight proteins in the nitrocellulose membranes and decreased protein binding19.

Figure 3

Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).

Sensitivity of LB

The sensitivity of the LB coupled with the identified optimal fixation method was further investigated. Serial dilutions of the pooled human serum proteins were separated by 10% SDS-PAGE, and the proteins were transferred onto PVDF membranes. The membranes were stained with lectins, Phaseolus vulgaris erythroagglutinin (PHA-E), LCA, Phaseolus vulgaris leucoagglutinin (PHA-L), and Aleuria aurantia lectin (AAL), combined with either no pre-treatment or with the identified optimal fixation method (Fig. 4). All types of lectins were shown to stain more glycoproteins when the analysis was combined with the fixation step (Fig. 4a, bottom panel) than without it (Fig. 4a, top panel), which was especially prominent for the glycoproteins with small molecular weight. These glycoproteins were not detected without prior fixation. Band intensities were analysed (Fig. 4b), and the sensitivity was shown to increase from 2.8- to approximately 15-fold when the method was coupled with the fixation step. Additionally, the ability of this fixation method in retention of protein when using nitrocellulose membranes, was determined, and shown to increase approximately from 3.7- to 12-fold (Supplementary Fig. 3).

Figure 4

Sensitivity of the LB method coupled with the fixation step, when using PVDF membranes. Indicated numerals are amounts (3.0, 1.5, 0.7, 0.3, and 0.1 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. (a) The blotted membranes were treated using the traditional (top panel) or optimised fixation protocol (bottom panel). (b) Quantification of band intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, sample fixation. The exposure times were the same for the same lectin blotting using fixation or no fixation. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. *Significantly different p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).

Applications of the optimised method in immunostaining and lectin staining

Immunoblotting and LB are used for the determination of protein expression and glycan level variations across different populations or conditions. CFTR, expressed in many epithelial tissues, is a key membrane protein in the complex network of molecules involved in epithelial ion transporters regulating epithelial permeability20. HIF-1, a heterodimer composed of a constitutively expressed HIF-1β subunit and a hypoxic response factor HIF-1α subunit, activates the transcription of genes that are related to critical aspects of cancer biology21. AFP is the only serum marker currently approved for clinical use in HCC diagnostics22. We not only analysed the expression levels of CFTR in HT-29 cells, HIF-1α in HEK-293T cells, GAPDH in liver tissue of mouse, and serum AFP in HCC patients, but also the glycosylation changes in the sera of prostate cancer patients, in combination with the optimised fixation method. In our analysis of CFTR expression in HT-29 cells (Fig. 5a), two bands were detected on membranes when blotting was performed either with or without the fixation step, which is consistent with a previous study23. However, for the visualisation of CFTR, the amount of total cellular protein required in no fixation group was 40 μg (Fig. 5a, top panel), while 10 μg was enough for visualisation in the optimised fixation group (Fig. 5a, bottom panel). In HIF-1α staining (Fig. 5b), the band was barely detectable even with 60 μg of protein sample in the no fixation group (Fig. 5b, top panel); in contrast, signal intensity was shown to increase eight-fold following sample fixation (Fig. 5b, bottom panel). We also tested tissue protein using this optimal fixation (Fig. 5c). The results showed that without the fixation step, 2.5 μg o tissue protein was required for the visualisation of GAPDH, while with the fixation step, the required amount was 1.3 μg of proteins, showing approximately two-fold increase in protein detection. For immunostaining of AFP (Fig. 5d), six and seven serum samples respectively obtained from healthy subjects and HCC patients were used. The increased AFP levels in the sera of the HCC patients were not detected using conventional procedure with 10 μg of total serum proteins (Fig. 5d, top panel). On the other hand, using the same amount of total protein, AFP expression was detected following the application of the fixation step (Fig. 5d, bottom panel). The elevation of serum AFP in HCC observed in our study was consistent with a previous report22.

Figure 5

Application of the optimised immunostaining and lectin staining methods. (a) CFTR levels in HT-29 cells. (b) HIF-1α levels in HEK-293T cells. (c) GAPDH levels in liver tissue of mouse. Various amounts (quantity represented in μg) of total cellular proteins analysed using 8% SDS-PAGE and immunostained using PVDF membrane and treated with or without fixation treatments. (d) AFP levels in the sera of healthy volunteers (n = 6) and HCC patients (n = 7), with different sample volumes using the PVDF membranes, with or without the fixation. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars). (e) AAL and PHA-E staining, using 6 μg of proteins from the sera of healthy volunteers (n = 6) and prostate cancer patients (PC, n = 10), blotted on PVDF membranes, with or without fixation. Three representative healthy samples (lane i, ii, iii) and seven representative prostate cancer samples (lane iv-x) are presented (left). Boxplot provides the quantification of the total band intensities (right). Circle, healthy subjects; square, prostate cancer patients. Student’s t-test. **P < 0.01 and ****P < 0.0001, healthy subjects vs. PC patients; #P < 0.0001, No fixation vs fixation groups. Band intensities were compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6.

Additionally, AAL and PHA-E lectins were used to analyse altered glycosylation in prostate cancer (PC) patients (Fig. 5e). The levels of fucosylated proteins and glycoproteins with bisecting glycosylation were significantly increased in the sera of prostate cancer patients, in comparison with those in the healthy subjects (P < 0.01), in both no fixation and fixation groups. The results obtained using the AAL blotting are consistent with those previously described for prostate cancer patients24. Furthermore, in both the AAL and PHA-E staining, the application of fixation led to detection of a larger number of bands than that obtained without fixation, allowing a more precise analysis of the differences between healthy and prostate cancer patient samples. The fucosylated protein (43 kDa) (framed in blue box in AAL staining) was detected in the sera of prostate cancer patients only following application of the fixation step combined with staining for AAL. In the PHA-E staining, a 40-kDa protein (framed in blue box) was detected only in the sera of the prostate cancer patients when no fixation procedure was applied, while it was detected in the sera of both healthy subjects and prostate cancer patients following the application of the optimised fixation step.

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小分子php蛋白,小分子蛋白的WB条件摸索_findtea的博客-CSDN博客

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小分子php蛋白,小分子蛋白的WB条件摸索 转载

2021-03-19 04:06:47

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小分子蛋白做WB有一些讲究,做了一段时间10-20KD的WB实验,有点体会,录于此备忘。并请各位老师批评指正。

WB的常规流程略。

小分子蛋白要用高浓度的分离胶,比如15%。

电泳我用恒压,电压太大会导致跑在最前面的小分子蛋白成锯齿状,70mV*30min浓缩胶,90mV*40min。另外,溴酚蓝跑到距胶底1cm以上,可分出目标蛋白即可,不必到底。

转膜我用湿转,恒流,电流太大或时间太长易转过,膜后再贴一张膜可检验,证实。感觉150mA*(30-40min)尚可。

转膜前的平衡时间短点,几分钟甚至1min就好。平衡所用的液体及转膜液成份相同,都不要加SDS,效果是天壤之别。

其它的WB相关细节(不限于小分子)还有:

脱脂奶粉封闭走个过场就行了,5-10分钟。

尽量用脱酯奶粉TBS液配一抗,自封口袋孵4度过夜,感觉比抗体孵育盒效果好,缺点是浪费抗体(但可回收啊)

二抗40分钟即可,摇不摇随便。

配分离胶时加超纯水,压气泡, 动作宜轻柔舒缓,尽量减少对分离胶的冲击,避免分离胶成分改变.

如果加样时稍稍离心,可使蛋白成分相对纯净,曝光时条带形态更好一点。

玻璃板烘箱烘烤后不能立即灌胶,如果不预冷至室温,将导致分离胶内明显气泡,这个有切身体会。另外,从冰箱内取出的凝胶试剂盒配制的胶据说也不能明显低于室温,需略等其升温,否则也易产生气泡。

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Zotero搭配Sci-Hub,真香! – 知乎

Zotero搭配Sci-Hub,真香!

由于之前在推文更新版|Zotero搭配Sci-Hub,真香!中使用的Sci-Hub的.se近期挂掉了,因此有必要对本文进行更新下。

目前(截止2020.04.15),可用的Sci-Hub域名有以下几个:

如果你想及时获取Sci-Hub最新可用网址,可以到青柠学术的GitHub网站[1]查看,我已经帮大家汇总好了。

我近期在Zotero中使用的是.ren域名,理论上以上四个都可以使用,大家可以自行更换为其中一个。

顺便把我的完整代码贴出来:

{
    "name":"Sci-Hub",
    "method":"GET",
    "url":"https://sci-hub.ren/{doi}",
    "mode":"html",
    "selector":"#pdf",
    "attribute":"src",
    "automatic":true
}

这里再提醒一下:

“ 如果你没有使用任何代理,由于网络环境问题,可能会有少部分从Sci-Hub下载的PDF出现损坏的情况,这时你可以考虑将上述代码中的https改为http,或者更换其他可用的域名,情况可能会好转。另外,可以逛逛本文在知乎的评论区[2],讨论的人还蛮多,说不从会有其他网友贡献更多好的办法。

除了改动部分,以下内容保持和之前的一致。


Sci-Hub有多香大家都知道!

Zotero有多香,你看了我的教程就知道了!

Zotero ,打造最佳文献生态(合集)

那要是Zotero+Sci-Hub,岂不是无敌了!

今天就教大家在Zotero内集成Sci-Hub,实现在Zotero中免费下载99%的文献!

从Zotero PDF retrieval谈起

从Zotero 5.0.56版本开始,Zotero迎来了PDF retrieval功能。详情可见Zotero官网的文章“Improved PDF retrieval with Unpaywall integration”[3]

该功能会在你用Zotero Connector保存文献时,自动检查Unpaywall上是否有可供下载的免费文献。

“ Unpaywall能免费下载文献,但你不要以为它和Sci-Hub一样是非法的。其实Unpaywall是个非盈利性合法组织,它整合了数千个Open Access期刊或数据库,将免费文献集中之后开放API,从而供其他平台使用。

假如你在网页端保存的文献是Open Access的,Zotero Connector就会将PDF同文献条目一起抓取,比如下面这样。

当然,对于已经在Zotero中却还没有PDF附件的文献条目,点击右键菜单中的Find Available PDF,即可下载文献,比如下面这样。

但是,毕竟Unpaywall只支持OA文献,而OA文献又只是少数。也就是说,通过Unpaywall无法解决付费文献的下载问题。

不过幸运的是,作为一款开源软件,Zotero的开发者为很多功能带来了可定制的能力,方便用户根据自己的喜好自定义。

PDF retrieval功能也不例外,Zotero允许用户自定义PDF解析器(custom PDF resolvers),也就是说你可以将其他网站作为PDF解析器,来替代Unpaywall。

详情可以访问Zotero官网链接Custom PDF Resolvers[4]

这为我们将Sci-Hub作为PDF resolver带来可能!

考虑到PDF resolver是内置在Zotero中的,这能保证我们能稳定使用该功能,就算Zotero更新了也丝毫不用担心,这一点就比使用第三方插件要有保障得多!

下面具体介绍如何将Sci-Hub作为PDF解析器!

设置Sci-Hub作为PDF解析器

PDF resolvers的设置在Zotero的Config Editor中。

我们打开Zotero的首选项,进入Advanced-->Config Editor

搜索extensions.zotero.findPDFs.resolvers,如下。

双击extensions.zotero.findPDFs.resolvers,默认情况下是只有一对[]

删除[],并将以下代码粘贴进去。

{
    "name":"Sci-Hub",
    "method":"GET",
    "url":"https://sci-hub.ren/{doi}",
    "mode":"html",
    "selector":"#pdf",
    "attribute":"src",
    "automatic":true
}

然后点击OK。

到此就成功将Sci-Hub配置为PDF解析器了,也就是说替代了默认的Unpaywall。

现在,无需重启Zotero,即可调用Sci-Hub免费下载文献了。

这里顺便提三点:

  1. 1. "url":"https://sci-hub.ren/{doi}"中,目前可用的域名有.tw.ren.si.shop,大家可以挑选其中一个,哪个用起来体验更好就用哪个。(当然,由于Sci-Hub经常更换域名,保不准改天哪个域名就挂了,或者有新的域名出来,因此此处的代码未来也会根据需要进行更新)
  2. 2. "url":"https://sci-hub.ren/{doi}"还能看到一点。由于Sci-Hub是通过doi下载文献的,因此该PDF解析器也需要doi。也就说你的文献必须要有doi,如果doi是空缺的,便无法通过PDF解析器免费下载文献。幸运的是,对于缺失doi的文献,我们可以通过插件zotero-shortdoi[5]插件一键抓取doi(参考文章zotero-shortdoi + Sci-Hub,让99%的文献都能被免费下载!)。
  3. 3. "automatic":true,如果设置为true,Zotero会自动下载保存到Zotero中的文献的PDF。比如你用Zotero Connector保存了一些文献到Zotero,它便会自动帮你从Sci-Hub下载文献,并附在相应文献条目下。如果你不需要自动下载,可以设置为"automatic":false

使用方法前面介绍过,主要有两种:

第一种:Zotero Connector

通过Zotero Connector保存的文献,会自动下载PDF,无需任何操作。(看不到进度条,下载速度取决于网速)

第二种:Find Available PDF

选中单篇或者多篇文献,手动点击右键菜单中的Find Available PDF,会弹出单独的窗口显示下载进度。同样,下载速度取决于网络速度。

关于下载速度取决于网络速度有下面两点需要注意;

  • 如果你未开启任何网络加速器(比如梯z),即正常使用网络,可以认为Find Available PDF的进度接近你手动从Sci-Hub下载文献的速度。大家应该都体验过,不开启加速器的情况下,Sci-Hub的访问速度还是比较慢的,甚至有时候PDF加载不出来。
  • 假如你开启了加速器,推荐使用全局代理模式,而不是PAC模式,因为两种情况下Find Available PDF的进度差异比较大(当然如果你不介意下载速度,使用PAC模式也是可以的)。不过提醒一下,下载完文献,记得切回到PAC模式,因为全局模式下Zotero无法同步文献到坚果云。

到此,本文就介绍完了!

可以看到,搭配Sci-Hub后,Zotero变得更加完美了!这就是开源软件的魅力,它能带来无限的想象空间。

如果你在使用中有什么问题,欢迎留言讨论!

相关链接

[1]

Sci-Hub最新可用网址查询: https://iseex.github.io/scihub/

[2]

Zotero + Sci-Hub知乎评论区: https://zhuanlan.zhihu.com/p/112141757

[3]

Improved PDF retrieval with Unpaywall integration: https://www.zotero.org/blog/improved-pdf-retrieval-with-unpaywall-integration/

[4]

Custom PDF Resolvers: https://www.zotero.org/support/kb/custom_pdf_resolvers

[5]

zotero-shortdoi: https://github.com/bwiernik/zotero-shortdoi/releases

这是尾巴

读过本文,如果觉得有收获,欢迎点赞转发收藏

编辑于 2020-04-18 11:32

文章被以下专栏收录

107 条评论

写下你的评论…

  • 智剑02-18

    最近我用这个,也存在找不到,然后我用doi去sci-hub.se找的时候发现几个问题,一是用short Doi找不到,但我用long Doi,可以在scihub找到文献,二是,利用zotero-shortdoi获取 long doi,然后zotero配置的sci-hub搜索引擎(doi)去搜索,发现找不到,观察之后是doi中的斜杠符号/发生了变化,变成%加两个字母,所以scihub就找不到,这长短doi的差别,及符号的改变,不知是什么原因,能请大佬指点一下,解决问题吗?

  • Shaun abc回复智剑03-07

    你好,请问下斜杠符号/发生转换的问题目前解决了吗,我也是在使用sci-hub搜索引擎是发现了这个问题

  • 知乎用户iiwR8p (作者) 2020-03-11

    这个我知道,最新的0.06版还是我前几天在Twitter 联系开发者ethan更新的,修复了不少bug。上一个版本0.05还是19年早期发布的,有不少问题。不过我不是特别推荐使用这种方式,会有一些人机验证弹窗等问题。我本文介绍的方法能实现在后台自己下载文献,不会对我们产生干扰。当然,感兴趣的都可以自行尝试下。

  • 滏阳河边捉蚯蚓回复知乎用户iiwR8p (作者) 2020-03-11

    每个域名下多了都有弹窗,并不是设置了 resolver 弹窗问题就解决了。

    真正没弹窗的是 scihub 的tg机器人。

  • 知乎用户iiwR8p (作者) 回复滏阳河边捉蚯蚓2020-03-11

    域名的流量控制肯定是有的。只是插件版的弹窗仅仅是个提示,无法直接人机验证,需要自己到scihub网页版输入验证码,解除人机验证。

  • BaoW2020-03-17

    你好,谢谢分享。但是按照你的方法,我右键点击“找到可用的PDF”时,依然显示无法找到PDF,但是我在浏览器中下载就可以下载,开代理和不开代理都试过,PAC和全局也试过,使用http://sci-hub.tw和.ren也试过,把https改为http也试过,都不行。请问我什么地方没有弄对吗?

  • 五十弦回复BaoW2020-03-25

    我也遇上了这种问题,不知道是哪里出了问题

  • BaoW回复五十弦2020-03-25

    我后面弄了几次,应该是需要输入验证码的问题,感觉这个插件还需要改进,目前能不能用可能看运气。我还是用网站下载的

  • 医路同行2020-03-24

    请问ZOTERO和MENDELEY在体验上各有什么优势?谢谢!
  • 叶落无声2020-03-19

    如果笔记本用的是windows的,那ipad又只能用ios版的,软件数据能互通同步吗?

  • 吸气牛2020-07-17

    非常好用,谢谢分享

  • 郝元斌2020-06-18

    非常有用的功能,谢谢。目前遇到一个问题,在下载一些 图书章节 的条目类型时,这类条目是没有DOI这一项的,但是会在URL 和其他项里有DOI。也能在sic-hub上下载到。请问能否加一个判断,在没有DOI项目的时候,在 其他 或者URL中找DOI信息?

  • sky2020-06-04

    您好,我想请教两个问题
    一是scihub每次抓取都会提示我Zotero Selector,然后要选择再确认才可以抓取,但是其他网页不会,怎么把这个提示给去掉呢?
    二是总是弹出输入验证码的提示,怎么让它不弹出来呢
    谢谢
  • TPOB2020-05-26

    (好用好用

  • 云浪2020-04-12

    那arvix得文章好像就不能自动下载了
  • 马生2020-04-03

    那个string value里面的url的值可以多值吗?比如多输几个备用域名之类的,就不用每次失效自己去改了

  • Mike Pan2020-04-01

    我靠,妙啊,你可真是个小天才
  • 盐选2020-03-28

    我在简书上看过这个。挺有用。可以搭配pop

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Tech Tips | In search of low molecular weight proteins | Proteintech Group

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  • Tech Tips | In search of low molecular weight proteins

Tech Tips | In search of low molecular weight proteins

Western Blot tips for low molecular weight proteins

Regular SDS-PAGE and Western blotting techniques are robust tools for investigating a broad spectrum of proteins with molecular weights (MWs) ranging from about 30 kilodaltons (kDa) up to 250 kDa. However, at extreme ends of the MW spectrum such techniques suffer from limitations: poor separation, signal reduction or even a total absence of target bands. Low MW proteins – generally any protein with a MW less than 20 kDa – are vulnerable to poor resolution and poor retention. Therefore they are challenging to detect with standard SDS-PAGE and Western blotting protocols…but they are not impossible to detect, period. Several protocol modifications can be employed when handling these MW featherweights for their retention and improved resolution at SDS-PAGE and subsequent Western blotting.

Try Tricine

The ‘standard’ polyacrylamide gels referred to above are uniform gradient glycine-Tris gel (which will simply be referred to as glycine gels). In general, glycine gels are ideal for resolving any proteins that fall within the range mentioned previously (30-250 kDas), given the total percentage of acrylamide mixture (T%) is adjusted accordingly. (Around 8% is used to obtain the best resolution of the higher end of this range, increasing up to 16%, or even 18%, for the lower end.) Acrylamide gels based on a Tris‑Tricine buffer system, however, will greatly improve your chances of ‘seeing’ your target band(s) if you are working below the 30 kDa range. Where you see a single band hovering around ambiguously near the expected MW on a glycine gel, you will see several individual, well-resolved bands at distinct MWs on a Tricine-based one. (These will be referred to as Tricine gels from this point on.) This difference is observed even when the pH and T% of the acrylamide mixture is the same within each gel (i.e. a 15% glycine gel versus a 15% Tricine gel at the usual pH values between 6.8 and 8.8).

You can find a recipe for a 15% Tricine gel in Proteintech’s Complete guide to Western Blotting

Comparison of Tricine- (A) and glycine-SDS-PAGE (B) separation of myoglobin fragments. Adapted from Schägger and von Jagow 1987.

The difference in separation capabilities of glycine and Tricine gels is attributed to the strongly differing properties of the glycine and Tricine compounds, such as their pK values and ionic mobility. We won’t go into too much detail here, but a basic explanation of why Tricine is more optimal for the separation of low MW proteins is that it ‘stacks’ proteins differently to glycine. The purpose of stacking layer is to pack proteins into uniform bands, so that groups of proteins enter the separating layer at the same time – like runners lining up at a start line before a race. (For instance, smeared bands can occur when a protein gets a head start from its protein ‘pack’.) A Tricine-based stacking layer shifts the upper stacking limit (i.e. the molecular mass of the largest protein in a given stack) down to as low as 30 kDa for the first stack1. Because proteins above the 30 kDa mark are already separated from the stack of sub-30 kDa proteins before reaching the separating layer, it prevents overloading at the interface between the gel layers, giving the stack containing the smallest proteins the best start to separationref1. The greater ionic mobility of Tricine also means that lower percentages of acrylamide can be used in gels to achieve the same degrees of separation – so higher, yet still moderate acrylamide concentrations can be used to resolve a narrow window of low MW proteins e.g. a 15% Tricine gel for the range between 5 and 20 kDa. But what about anything below 5 kDa you ask? Adding 6 M urea in the gel mixture enhances the resolution of small proteins furtherref2, which is recommended for anything lower than 5 kDa.

Protein transfer

As well as ensuring the optimum separation of low MW proteins, you also need to take care at the protein transfer stage. Low MW proteins are susceptible to ‘over transfer’ – loss of sample due to lack of retention by the transfer membrane and/or too rapid transfer.

Membrane choice and pore size

Most labs have a preferred choice of Western blotting membrane, be it nitrocellulose or PVDF (polyvinylidene fluoride), but PVDF is a better choice in the case of smaller, low MW proteins. It has a greater capacity to bind proteins compared to its nitrocellulose counterpart. PVDF has a protein binding capacity of 170 to 200 μg/cm2 while nitrocellulose has a protein binding capacity of 80 to 100 μg/cm2 ref3This article discusses a few more of the considerations you should take into account when choosing a membrane for Western blotting, complete with images of the microstructure of both PVDF and nitrocellulose membranes (which is part of the explanation protein retention properties of PVDF are slightly higher).

Whichever membrane you choose, be aware there are various pore sizes on offer; both are available as 0.45 μm, 0.2 μm, or 0.1 μm versions. Opt for a smaller pore sized version to obtain better transfers of your low MW target proteins – a 0.2 μm membrane is more than adequate for use with any proteins weighing in less than 20 kDa. The Proteintech lab uses Millipore’s Immobilon PSQ PVDF membrane for the transfer of low MW proteins.

Transfer conditions

In addition to membrane choice, other factors like transfer system, length, temperature and buffer composition come into play when dealing with low MW proteins. Semi-dry transfer systems seem to have the edge over wet transfer apparatus in this arena (probably due to the simple fact that semi-dry systems are less vulnerable to over transfer because of their efficient transfer times). Be aware however, that semi-dry transfer systems can suffer from reproducibility issues (but this should be less so with low MW targets).

In the case of small proteins, less is often more when it comes to transfer length and voltage (or amperage depending on your setup). How much you should adjust these by will be down to the brand and model of system being used. Look up the recommended transfer times and voltages for proteins below <30 kDa in the equipment manual – if one of these isn’t available, you should still be able to find this information on the manufacturer’s website.

Other tips

You may be using bromophenol blue in the sample loading buffer when running samples on a gel, but be aware some proteins will escape the dye front!

You can choose to soak your gel in an SDS-free buffer (or simply good old H2O) for 5 minutes prior to setting up your transfer. This helps to remove SDS, which coats small proteins and protein fragments in negative charges, increasing their rate of passage through the Western blot membrane.

Happy hunting!

References

1. H Schägger and G von Jagow. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166(2):368-79.

2. H Schägger, Tricine-SDS-PAGE, Nature Protoc. 2006;1(1):16-22.

3. The Protein Man’s Blog, PVDF or Nitrocellulose – Which Membrane is Best? Nov 12, 2014:http://info.gbiosciences.com/blog/bid/203026/PVDF-or-Nitrocellulose-Which-Membrane-is-Best


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VITA比色新软件—颜色传达的创新工具-丰达123的博客-KQ88口腔博客

VITA比色新软件—颜色传达的创新工具-丰达123的博客-KQ88口腔博客

VITA比色新软件—颜色传达的创新工具

发布时间:2013-07-23 08:26  | 文章分类:综合浏览次数:185  |  评论:0  | 推荐数:0
 

把牙齿颜色的所有相关信息无损失地传达是对于牙科修复体制作的正确复制和材料选择的决定性因素。 鉴于这个原因 VITA Zahnfabrik 为最佳颜色传达开发了最新软件 VITA 比色辅助软件

摄影诊断

从2009年秋季从www.shadeassist.de上可免费下载基础版本。

运用 VITA 比色辅助软件(XP, Vista),牙科诊断可简单的记录,处理和为发送给牙科技师或病人做准备。它可以被组成任何数量的单独病人档案并打印用VITAPAN16色经典比色板 或 VITA 3D-MASTER 线性比色板比色的结果, 用VITA 无线电脑比色仪测定的结果, 与描述牙外形诊断相关的,VITA比色辅助软件协助编辑文案评论和数码照片和图表。

个体诊断的报道总结使以集成文件夹方式得到快速的治疗进程的浏览成为可能。.在“帮助”菜单下面详细介绍了软件的功能(有德语和英语)。

创造一个新的报告

作者:丰达牙科 http://tesco-dental.com

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以上网友发言只代表其个人观点不代表口腔医学网的观点或立场

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    申明:本博客原创文章归作者本人和KQ88口腔博客(牙医文集)共同所有,如有转载或引用文章请与原作者联系,不得私自转载!
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    转录因子的靶基因,看这一个数据库就够——Harmonizonme – 王进的个人网站

    转录因子的靶基因,看这一个数据库就够——Harmonizonme

    2021-12-142021-12-14

    对于转录因子而言,我们最想知道的信息就是其对应的靶基因。转录因子相关数据库非常的多,有些数据库直接提供了靶基因的信息,比如TRANSFAC, 有些数据库只提供了motif的信息,比如JASPAR, 我们只能通过软件预测在基因的启动子序列上是否有对应的motif, 从而识别转录因子的靶基因。

    TRANSFAC收费,而JASPAR的靶基因预测又需要一定的生信技能,有没有哪个数据库既免费,又直接提供了靶基因数据呢?

    这种数据库肯定是存在的,比如之前介绍过的TRRUST等数据库,但是本文的主角是另外一个数据库,Harmonizonme。

    将各个Resource来源数据库中的原始信息加以整理,得到更加直观,方便使用的数据集Datasets,然后将所有的整理好的信息存储在同一个数据库中,就得到了Harmonizonme数据库,网址如下

    http://amp.pharm.mssm.edu/Harmonizome/

    目前该数据库包含来自66个Resources数据库,114个数据集datasets, 共有57620个基因,295496个属性,71927784个相互作用关系。分类情况如下

    可以看到有非常多种类型的信息,本文只介绍转录因子的靶基因数据集,在官网首页,根据关键词检索相关的数据集,示意如下

    最终可以检索到以下6个转录因子靶基因数据集

    以TRANSFAC Curated Transcription Factor Targets数据集为例,链接如下

    http://amp.pharm.mssm.edu/Harmonizome/dataset/TRANSFAC+Curated+Transcription+Factor+Targets

    该数据集是手工收集整理的转录因子靶基因数据集,示意如下

    共包括201个转录因子的靶基因信息,点击每个转录因子,可以看到相关的靶基因,示意如下

    除了在线浏览特定转录因子的靶基因外,还可以方便的下载该数据集,点击下图中红色方框标识的文件进行下载即可

    该文件列数很多,前几列示意如下

    source

    source_desc

    source_id

    target

    target_desc

    target_id

    SLC16A9

    na

    220963

    ALX1

    na

    8092

    SLC26A3

    na

    1811

    ALX1

    na

    8092

    ABHD17B

    na

    51104

    ALX1

    na

    8092

    这里的id指的是基因对应的Entrez Id, source值的是靶基因,`target值的是转录因子,通过该数据库可以方便的得到转录因子和靶基因之间的调控关系。

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    转录调控必知数据库:ENCODE – 云+社区 – 腾讯云

    转录调控必知数据库:ENCODE

    发布于2020-10-27 16:42:58阅读 1K0

    之前我们在介绍很多转录调控相关的数据库的时候,都会提到这些数据库包含了ENCODE数据库。那么ENCODE数据库是什么样的数据库呢?

    ENCODE

    (Encyclopedia of DNA Elements, https://www.encodeproject.org/),翻译成中文就是DNA元素百科全书,其主要目的是为了了解这个基因组当中的调控反应,主要方法还是利用高通量的测序技术来进行分析的。

    按照上图的展示,目前的ENCODE通过多种测序数据来反应基因组变化的过程,分别是通过

    • Hi-C 来观察三维基因组
    • ATAC-seq/chip-seq 研究基因的转录调控
    • 甲基化芯片来研究甲基化的调控作用
    • RNA-seq 来研究基因表达的变化
    • RIP-seq 研究在转录后调控的信息

    我们可以通过ENCODE数据库来检索自己想要的数据。类似很多转录调控数据库也是在ENCODE数据库获得目标原始数据后,进行分析后构建的自己数据库。

    数据统计

    目前ENCODE数据不止是包括人的数据,现在包含了四种物种的数据,主要含有: 人、老鼠、蠕虫、苍蝇这四个物种。

    我们可以点击相关的数据类型,就可以得到ENCODE数据的这个类型的所有数据了。例如我们点击: DNA binding即可看到数据库的所有数据。

    数据检索

    同样的,我们可以基于自己的目的来检索想要的数据。

    这里我们检索: CTCF。就可以看到和CTCF相关的数据集了。其中前四个是不同物种chip-seq的数据。

    我们可以选择 CTCF(Homo sapiens),就可以看到具体的在人的物种当中所有和CTCF有关的数据集了。这里会显示不同的组织的数据,我们可以选择想要查看的组织类型进行查看。

    具体数据集介绍

    对于不同的检索方式,我们都能到具体数据集内容介绍里面。对于数据介绍基本格式基因相同,这里我们就用:ENCSR331OGX这个CTCF相关的chip-seq数据来简单介绍一下。

    1. 数据汇总信息。这里我们能看到数据集基本信息,包括患者基本信息。对于ENCODE的数据,都会放到GEO里面,所以我们在GEO里面其实也是可以检索到ENCODE的数据的。
    1. 具体的数据文件。这里我们可以看到数据的所有原始数据,包括测序数据的fastq数据以及基于ENCODE分析流程分析的所有bam文件和peak文件。

    对于数据的peak文件,可以通过基因浏览器来进行查看。我们之前介绍过一个好看的基因浏览器。ENCODE默认的是UCSC的基因浏览器,可以点击 Visualize来进行查看。

    1. 数据处理流程:ENCODE提供了关于数据的标准处理流程,如果要使用他们的数据结果的时候,可以知道是怎么处理的;同时如果我们有自己的数据的话,不知道怎么处理,也可以参考这个数据处理流程的。

    数据库总结

    关于ENCODE基本介绍就是这些的。这个数据库主要还是一个偏向于原始数据储存的数据库。我们如果需要进行原始数据分析的话,可以从这个下载数据。但是如果是想要直接检索转录调控的结果的话,可以使用一些基于ENCODE数据分析完的数据库例如:我们之前介绍的Chea3[数据库推荐]多基因转录因子调控网络预测或者Cistrome等只要提到ENCODE数据的这些转录因子调控数据库。

    建议还是如果要进行课题设计,可以使用那些对ENCODE加工的数据库好一些,这样只需要检索就可以获得结果。如果想要自定义的分析,那还是下载原始数据好一些,不过这个对于分析能力的要求就要高一些了。

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