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【求助】在研究某个蛋白进入细胞核情况
在研究某个蛋白进入细胞核情况的时候,文章中有人对蛋白进行免疫荧光后,用激光共聚焦显微镜观察,后又用VTIHCS软件进行分析。还有人分别提取核蛋白和质蛋白,用western blot检测该蛋白的变化水平。也有人同时进行了上述的实验。同样是看蛋白的定位,上述几种做法的差距在哪儿,意义是什么呢?还望给位大侠赐教!【求助】在研究某个蛋白进入细胞核情况
查看完整版本请点击这里:
【求助】在研究某个蛋白进入细胞核情况
【求助】在研究某个蛋白进入细胞核情况
最新回复
jishiben (2015-11-15 18:38:03)
直接用电子显微镜观察或冷冻电镜观测,只能杀死细胞,不能实时观测。
分别提取核蛋白和质蛋白,用western blot检测该蛋白的变化水平,我才不去做呢!费时费力,做蛋白质的表达量用分子筛层析即可,或者用质谱直接检测出不同蛋白质的表达量更省事!定位用第一段的,省事省力,而且原位观测和活细胞观测!分别提取核蛋白和质蛋白,用western blot检测该蛋白的变化水平,这样费力费时的活,谁愿意干就去干!反正我不去当这样的低效劳动力!
就这样!
danzi (2015-11-15 18:38:27)
xgy412 (2015-11-15 18:38:48)
wwwh (2015-11-15 18:39:09)
8899 (2015-11-15 18:39:49)
言归正传!
生物芯片的基本原理可能有三:
1.把生物大分子或可能结合生物大分子的有机小分子蚀刻在硅片表面:可能结合生物大分子的有机小分子有必要游离出来羟基,磷酸基,氨基,羧基等蛋白质的侧链上的相关基团连接成共价键;羧基等连接成共价键,这些基团与直接把生物大分子在硅片表面时,这需要硅片表面的羟基与蛋白质的侧链上的羟基,羧基等连接成共价键。
2.点阵列原理:每一个硅片表面的生物大分子(搜索ssDNA,RNA,抗体等)都在硅片表面有特定的精确的位点,并且能够为计算机直接识别,在其与外来生物大分子相互作用的前后皆是如此。
3.分子杂交原理,可能叫分子相互作用更好些。即每一个硅片表面的生物大分子(搜索ssDNA,RNA,抗体等),在其与外来的某些生物大分子相互作用的前后皆是如此,并且能够有某种标记物能够为高通量的光学检测仪器和计算机直接识别。
生物芯片之一是蛋白质芯片,主要是抗体蚀刻在硅片上。用蚀刻某种核蛋白的克隆抗体的硅片(蛋白质芯片)接触缓冲溶液中的和提取液扥点样,不仅能够识别出该种核蛋白的存在而且能够检测出其表达量。
质谱也是一样,识别出该种核蛋白在核内的存在而且能够检测出其表达量。
hcy517 (2015-11-15 18:40:31)
对于细胞核用蛋白质组学方法也可以,当然只有一个目的蛋白质要确定就直接走MS就是了,适当分离一下,不用分子筛,亲和之类细提纯,主要把细胞核分级离心出来,然后粉碎就行。如果该蛋白质比较难以分出来,或者为了MALDI-TOF-MS.测定那步比较轻松,可以用到一维电泳或者二维电泳分离可能的目的蛋白质。
下面主要我讲一下有关蛋白质组学的质谱数据库的搜索的算法原理的简介。
分别用MALDI-TOF-MS.测定了一系列可能是目的未知蛋白质的序列的某些有限酶解肽段或者全蛋白质的分子量(你的材料没说质谱测定的斑点回收物是什么以及怎么测定,我是按照一般用MALDI-TOF-MS.确定蛋白质的方法猜测的)可能是用有限酶水解制备的多肽片段,从蛋白质数据库中搜索蛋白质完全没有进行蛋白质的全序列测定-----除非是数据库中没有的全新蛋白质!
蛋白质组的质谱数据库搜索原理也有很多算法,我只举其中一种,有些代表性。一般蛋白质组的质谱数据库搜索原理第一步是将所得所测的肽质量数与蛋白质组的质谱数据库中的每一个蛋白质的理论肽谱进行比较。当计算值落在误差设定范围以内时,就记作一个匹配。与计算匹配的肽段数量不同,MOlecular Weight SEarh(MOWSE)使用经验因子来为每一个肽匹配设定一个“权重”。该权重因子矩阵在构建数据库时产生,方法是:
先产生一个频率因子矩阵F,在此矩阵中,每一行代表肽质量数相差100道尔顿,每一列代表蛋白质的质量数相差100kD道尔顿。当分析每一个序列时,设定合适的矩阵元素f(i,j)步长以便将肽质量的大小作为蛋白质的质量的函数进行统计分析。矩阵F中的每一列的元素除以该列中的最大值,从而使矩阵F归一化,并且得到MOlecular Weight SEarh(MOWSE)因子矩阵。在用肽质量实测值对理论肽质量数据库检索后,按下式计算每一次检索的应得的分数(score):
score=50000/,M(protein)是蛋白质的分子量,∏m(i,j)为MOlecular Weight SEarh(MOWSE)因子矩阵中的元素的乘积。
再举一个蛋白质组的质谱数据库搜索的例子:
1.“Algorithms and Software Tools for Identifying Proteins from ESI Tandem MS Data: Sequest
The firs t algorithm/program to identify proteins by matching MS-MS data to database sequences is Sequest, which was introduced by John Yates and Jimmy Eng in 1995. Several similar software tools Protein Identification with MS Data 101 have been introduced and these will be discussed below. However, Sequest will be described in greatest detail as representative of this class of tools. The value of programs such as Sequest is that they provide a relatively rapid assignment of MS-MS spectra to specific peptide sequences in databases. This allows fast reduction of large volumes of LC-MS-MS data in proteomics analyses. However, it is important to emphas ize that Sequest and similar programs do not actually perform de novo interpretation of the spectra per se .Consequently , the output of these programs depends on the quality of the MS-MS data obtained and the completeness and accuracy of the database used.
Here’s how Sequest works. When the MS instrument obtains an MS-MS scan, it not only records the MS-MS scan itself, but also the m/z value of th e precursor ion. This information is stored together
with the scan data. After the analysis is complete, the user sits at the computer and opens the Sequest program. The user then selects the data file containing the MS-MS scans to be analyzed. The user can tell Sequest what enzyme (e.g., trypsin) was used to digest the protein sample and also specifie s whether singly or doubly charged ions were subjected to MS-MS. Finally, the user selects a database against which the MS-MS data are to be compared.
Once the program starts, all of the proteins in the database are subjected to a virtual digestion with the enzyme specified by the user (e.g., trypsin) . This generates a master list of possible peptides for comparison to the MS-MS scans. Then each MS-MS scan is analyzed as follows:
• The precursor m/z for each MS-MS scan is used to select peptides from the database with the same mass (within a defined mass tolerance). If no digestion enzyme was specified, the program simply select s all possible peptide sequences that correspond to the mass of the peptide ion analyzed in that MS-MS scan.
• Theoretical MS-MS spectra are generated from each of the selected peptides.
• The MS-MS spectrum being analyzed is compared with each of the theoretical MS-MS spectra generated from the database.
• A correlation score is calculated for each match between the
MS-MS scan and the theoretical MS-MS spectra.”
2."Soft ware Tool s for Peptide Mass
Fingerprinting: Scoring the Results
In MALDI- TOF spectra from real samples, there are typically dozens of m/z signal s. Peptide mass fingerprinting software can usually match just about all of these to some entry in a database. However, given errors in m/z measurement, frequent sample contamination , and the presence of unanticipated posttranslational modifications, not all of the matches will point to the same proteins. So how do we score the hits to determine which protein best matches the data?
The simplest approach is to assign the highest score to proteins whose predicted tryptic peptides match the greatest number of m/z signals in the MS data. If we search only one m/z value, then several proteins could be equally good matches. However, as we search a greater number of m/z values, mo re matches correspond to a particular protein and lead to a greater score for that protein vs others. This fairly simple approach works reasonably well with very good MS data. However, it tends to assign higher scores to larger proteins.
As note d earlier, larger proteins yield more tryptic peptides, so the chances of a match to one of these is greater for larger proteins than for smaller proteins.
malong (2015-11-15 18:42:11)
hcy517 (2015-11-15 18:43:02)
基因序列比对是将者基因序列位点上的匹配位点(相同或者相似残基)与不匹配位点(不相似残基)按照一定的记分规则转化为序列间相似性或者差异性的数值来加以比较,相似性最大的比对结果具有最多的匹配位点,从数学上讲,应该是最优的比对结果;但是从数学模型或算法得出的最优结果在多大程度上反映了序列之间的相似性以及它们的生物学特征之间的关系,将取决于将生物学问题简化成数学问题的过程,而这一过程也是生物信息处理最难解决的问题。
rrra6 (2015-11-15 18:45:24)
兔子 (2015-11-15 18:46:18)
【求助】在研究某个蛋白进入细胞核情况