The pHEK293 Ultra Expression Vectors are plasmids for transient overexpression of a protein of interest in human HEK 293 cells. Protein expression using these vectors can be up to 10 times higher than what is obtained from vectors with conventional cytomegalovirus (CMV)-derived promoters. Prior to purchasing these products, all customers must complete a purchaser"s agreement to submit with the order.
High protein expression is accomplished using the HIV-1 TAR-Tat system.
The pHEK293 Ultra Expression Vectors are plasmids for transient overexpression of a protein of interest in human HEK 293 cells. Protein expression using these vectors can be up to 10 times higher than what is obtained from vectors with conventional cytomegalovirus (CMV)-derived promoters. Prior to purchasing these products, all customers must complete a purchaser"s agreement to submit with the order.
High protein expression is accomplished using the HIV-1 TAR-Tat system. The TAR (transactivation response) element is an RNA sequence derived from human immunodeficiency virus type 1 (HIV-1) that forms a stem-loop structure at the 5’ end of HIV-1 RNAs. Tat is an RNA-binding protein that activates transcription by binding to the TAR and promoting transcriptional elongation.
The pHEK293 Ultra Expression Vectors exploit this transcriptional activation mechanism; the TAR sequence is present in the 5" untranslated region of the target gene, and a Tat expression cassette is included either on the same vector (Cat. # 3390) or on a separate plasmid (Cat. # 3392). This system allows efficient expression of Tat protein, thereby greatly increasing the amount of target protein that is expressed. Since the TAR sequence is located in the untranslated region of the target gene, it does not affect the amino acid sequence of the expressed target protein.
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可以用移液器将细胞吸出来并高速离心，沉淀重悬于PBS中洗涤，接着就可以裂解提取蛋白了。可以用超声，酶解等等，裂解后离心收集上清。
做的比较好的，一般都是上海地区的，你可以看下基尔顿生物。
我刚开始做转染，悬浮细胞，分别做过表达和敲减，看了很多文献，大都没有提及转染后是用转染的这同一批细胞同时做pcr，wb，cck8，凋亡，细胞周期；还是说这次转染只做pcr或wb，再转染一次做cck8或细胞周期。剩下的功能试验均同前，转染一次做一次？我养的是悬浮细胞，转染后做cck8这些功能试验前需要离心换液吗？跪谢解答！
细胞状态与密度；转染试剂的类型、用量；DNA的品质、用量；转染复合物的品质、作用细胞的时间长度。
