Protein expression and purification Mammalian cells HeLa

Get tips on using pCX-puro-mouLH to perform Protein Expression Eukaryotic cells - CHO mouse LH

Get tips on using pCX-puro-mouFSH to perform Protein Expression Eukaryotic cells - CHO mouse FSH

Get tips on using pHT43-BMP2-D to perform Protein Expression Prokaryotic cells - B. subtilis rhBMP2

Get tips on using pHT43-BMP2-M to perform Protein Expression Prokaryotic cells - B. subtilis rhBMP2

Get tips on using pJL TRBO-G to perform Protein Expression Prokaryotic cells - A. tumefaciens GFP

Get tips on using pHR-CMV-TetO2-CD45 to perform Protein Expression Eukaryotic cells - HEK293 CD45

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

Get tips on using QuantiPro™ BCA Assay Kit to perform Protein quantification Mammalian cells - L929

Get tips on using QuantiPro™ BCA Assay Kit to perform Protein quantification Mammalian cells - RAW264.7

Get tips on using CelLytic™ NuCLEAR™ Extraction Kit to perform Protein isolation Mammalian cells - Human eutopic endometrial stromal cells