The ChIP Elute Kit uses a fast and simple method to reverse cross-link, elute, and purify DNA captured by chromatin immunoprecipitation (ChIP). This kit replaces long and tedious protocols for the final stages of ChIP experiments. Single-stranded DNA (ssDNA) can be recovered in approximately one hour (compared to traditional methods that may require overnight incubation) and is directly compatible with the DNA SMART ChIP-Seq Kit.
The ChIP Elute Kit uses a fast and simple method to reverse cross-link, elute, and purify DNA captured by chromatin immunoprecipitation (ChIP). This kit replaces long and tedious protocols for the final stages of ChIP experiments. Single-stranded DNA (ssDNA) can be recovered in approximately one hour (compared to traditional methods that may require overnight incubation) and is directly compatible with the DNA SMART ChIP-Seq Kit.The DNA SMART ChIP-Seq Kit adds Illumina-specific sequencing adapters to the ends of DNA fragments in a ligation-independent manner. Generating libraries for ChIP sequencing (ChIP-seq) can be difficult due to the small amounts of DNA recovered from ChIP experiments, particularly when using antibodies against low-abundance transcription factors. The DNA SMART ChIP-Seq Kit uses a novel version of SMART template-switching technology—adapted for DNA templates—to generate next-generation sequencing (NGS) libraries for Illumina platforms from 100 pg–10 ng of input DNA. Either ssDNA or double-stranded DNA (dsDNA) from ChIP experiments can be used directly with this kit, making it compatible with DNA recovered with the ChIP Elute Kit or by other methods. This highly sensitive kit generates robust sequencing libraries from low-input DNA samples, with minimal handling, in about four hours.
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第一，血清成分复杂，最好用密理博的Montage Albumin Deplete Kit去除血清里50%的白蛋白
第二，磷酸化蛋白提取蛋白的时候，最好加入原钒酸钠，和磷酸酶抑制剂，防止蛋白脱磷酸化。
第三，不要用牛奶做封闭剂，用BSA。因为牛奶中含有磷酸化酪蛋白，防止非特异带。
第四，如果条带杂带多，而且，条带弱，可以选择millipore的signal boost，增加条带特异性，而且，增强条带的亮度。
转膜后就要区分与胶接触的一面与另一面了。话说楼主好奇的话可以跑蛋白的试试看哈。比如目的蛋白的胶用光滑面贴着胶，内参蛋白用粗糙面贴着胶一起转膜试试转膜结果看看呢。
1. 使用预染 Marker，不过分子量不是特别准；
2. 所使用的 Marker 条带与待测蛋白质带有相同的抗原表位，比如都带有 His 融合标签；
3. 可以使用普通的 Marker 转膜使用 丽春红 等染色，然后在膜上标记出 Marker 各条带的位置。
考马斯亮兰染液也可以重复用。新配的染液10分钟即可，重复3次后要染30分钟。
一抗二抗可以重复利用,但是注意要在5%milk中加入0.2% sodium azide ,并且用完以后放入4度保存,我的经验重复使用4-5次是肯定没有问题的.如果保存不当,就会污染微生物,只能丢弃.
