Tri-peptide chloromethylketones have been utilized extensively to irreversibly inhibit various serine proteases (1-5). Among the most common chloromethylketones are FPRCK (Phe-Pro-Arg-chloromethylketone; commonly referred to as PPACK), which is a rapid thrombin inhibitor and EGRCK (Glu-Gly-Arg-chloromethylketone; commonly referred to as GGACK), which is a rapid factor Xa inhibitor (1). Both FPRCK and EGRCK are used extensively during protein isolation procedures to inhibit serine protease activity and prevent further conversion of zymogens to active enzymes. Recently, the modification of these tri-peptide chloromethylketones with reporting groups, such as fluorescent probes (6-8,14), radioactive labels (9) or thioreactive-labels (10), has provided a unique approach to the study of various serine proteases. These probes are useful because they allow a means of reporting molecular changes in an enzyme, and not its zymogen, while also inhibiting the enzymatic activity.
The use of biotin as a reporting group has been used extensively with antibodies in ELISA based assays and in western blotting. The biotin, in conjunction with avidin, creates a highly sensitive method for detecting antibodies, and therefore, antigens. By modifying the tripeptide-chloromethylketones with a biotin group, the sensitivity of the avidin/biotin system can be extended to study serine proteases without the need for specific antibodies to the active enzymes.
Biotinylated tripeptide chloromethyl ketones can be used in a variety of ways (11-13). First, the compounds can be reacted with unwanted serine proteases in a sample or preparation, and can then be removed along with the protease using avidin-Sepharose (11). Second, the biotinylated-serine protease can be visualized on a blot without the use of specific antibodies (11). Third, the biotinylated serine protease can be quantitated in an active-site specific immunoassay (12,13,15), such as the tPA-CASSIA (see Assay Kits). The spacer utilized on these compounds has been optimized to allow good reactivity of the biotinylated FPRCK and the biotinylated EGRCK in the above mentioned procedures.
In addition to biotinylated chloromethyl ketones, fluorescein labelled compounds are also available. The fluorescein labelled compounds are useful in both Western blot and fluorescent imaging applications.
Biotinylated and fluorescein labelled FPRCK and EGRCK are prepared by the method of Williams et al. (11).
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求问酶切体系为10ul,内切酶1ul,37℃水浴45min，不知道DNA产物是否完全被切开了？
所用内切酶信息如下
同一管dna有的酶可以切开，比如dra1，ecoR1,Hind111
有的酶又完全不能切动，如BamH1，sac1，xba1，xho1，pst1
请问高手这是什么原因？?
配后是否应该分装,因为不能反复冻融?
多谢!
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【】可以这样输入，智能ABC，输入“v1”，后翻3页，就可以看见了，或用紫光拼音中文输入法，输入方括号就是粗体的中括号，实在不行请复制本公告中【】
感谢赐教~！
我做的双酶切反应，酶分别是宝生物的BamHI和XhoI，双酶切体系是：载体及目的片段分别是30ul，通用buffer4ul，XhoI、BamHI各2ul，无核酶水2ul，总体积40ul。载体和目的片段都是经37度酶切6h。载体上面这两个酶切位点的距离是6个碱基，目的片段是由pcr反应获得的，从puc19中p出来的，两端带有这两种酶的酶切位点，酶切完成后，做连接反应，体系如下：目的片段（全长1441bp）4ul，无核酶水3ul，10*T4DNA连接酶缓冲液1ul，载体1ul，T4DNA连接酶1ul，总体积10ul。16度过夜。之后摇菌送沉菌测序结果回来，送了5管一个都不对。重复实验两次还是不对！
之后改为先用BanHI单切载体，体系如下：载体17ul，酶1ul，buffer2ul，总体积20ul，30度切6小时后，Promega纯化试剂盒直接纯化，完了之后再用XhoI，37度酶切6小时，体系如上，再次用promega纯化试剂盒纯化，目的片段用双酶切体系酶切，之后做连接反应，之后铺板，挑取菌落共10个，摇菌13小时，取菌液做pcr鉴定，结果10个菌落全是引物2聚体，跑出来的条带都是100bp左右。
各位老师这是怎么回事呢？为什么连接不上呢？小弟先谢过各位大哥了！
