chip实验误差分析

1、cross-link：甲醛浓度、胶联时间一般都使用1%formaldehyde，但也有人用浓度偏高的，比如： add 10 ml of Fresh PBS and add 270 ul of 37% formaldehyde, swirl gently to mix, and place at room temp 10 min. 胶联时间很是关键： The duration of formaldehyde treatment is a point of optimization. Shorter treatments will generally yield higher concentrations of soluble protein, but less extensive cross-linking of proteins to DNA. Longer treatments will yield the opposite, and consequently there will be less material in the extracts for doing IPs. However, this is not the first step that should be optimized. 20 minutes is probably a good starting point for this step unless the effect being observed is expected to occur in a very short time frame. In that case 5-10 minutes formaldehyde treatment may be more appropriate. Optimization of this step involves maximizing the IP/IN ratio for the protein-DNA interaction being assayed, and keeping the duration of formaldehyde treatment short. This can be examined by PCR after IP conditions have been optimized by Western analysis.所有的都需要自己来摸索。2、sonication：时间、强度不同的材料其打碎时间不一样，并且打碎程度还与胶联的时间有关，这一步的摸索比较复杂，需要耐心。假如做的是同一个材料，最好和胶联一起摸索条件，一旦摸索一个合适的，就不要更改了。The water bath sonicator develops a film over the membrane used to sonicate the samples. For best results run the sonicator at level 10 for a few seconds to break the film up before adding samples to the water and sonicating at level 5. It may also be a good idea to add some ice to the water bath to keep the samples as cool as possible.打碎后要进行解胶联（reverse cross-linking）以检测打碎的程度，按照常规步骤比较繁琐，以下是一个快速的检测解胶联的方法：Quality Control: After the extracts are made you can verify efficient shearing of DNA by the following:1. mix 20 ul extract + 1ul 10% SDS + 0.5 ul proteinaseK2. incubate 1hr at 37 degrees C followed by 2 hrs at 65 degrees C3. phenol/chloroform extract the samples4. chloroform extract the samples5. EtOH ppt DNA with 10 ug/ml glycogen (final concentration)6. resuspend DNA in 20 ul TE7. treat with 40 ug/ml RNAse for 1hr at 37 degrees C8. run half of each sample on a 1.5% agarose gel with EtBr with an appropriate DNA sizing ladder (sheared DNA should run between 100 bp and 1000 bp)3、抗体的选择：一般来讲，如果做组蛋白的chip，有商业化的抗体可以买，比如ABCAM，UPstate等，但假如是非商业化的转录因子（或其它）的话，就要自己摸索条件了。给大家一个建议：The amount of Ab used in the assay must be determined empirically. Important considerations include: the fraction of your protein-of-interest the Ab is able to pull down, how much the protein is left after elution, and the ratio of specific PCR signal to noise. Before proceeding to the main experiment it is important to optimize the IP conditions using a combination of Western and PCR analyses to address the aforementioned issues.需要注意的地方：不要着急，我会慢慢的修改的 wendysun edited on 2006-06-27 15:16 2006-06-25 13:55 分享到那里 复制网址 新浪微博 豆瓣社区 腾讯微博 开心网 人人网crosslink的原理Fig. 1. Cross-linking reactions with formaldehyde and bifunctional imidoester reagents. The figure depicts the chemical cross-linking reactions mediated by formaldehyde and bifunctional imidoester reagents. The formaldehyde reaction proceeds through initial formation of a Schiff base that subsequently condenses with a second amine to form the cross-linked product. The imidoester cross-linking reaction proceeds through two subsequent (or simultaneous) reactions of an imidoester group with a primary amine (for example the e amino group of lysine) at alkaline pH to yield an amidine product. 原理 2FIG. 2. Chemical cross-linking of DNA and proteins by formaldehyde. Formaldehyde (HCHO) is a very reactive dipolar compound in which the carbon atom is the nucleophilic center. Amino and imino groups of proteins (e.g., the side chains of lysine and arginine) and of nucleic acids (e.g., cytosine) react with formaldehyde, leading to the formation of a Schiff base (reaction I). This intermediate can react with a second amino group (reaction II) and condenses. Cross-links may be reversed by heating in TrisHCl-containing buffers. This leads to a drop in pH and protonation of amino groups,