Protein isolation Bacteria

Get tips on using Host Cell Residual DNA contamination LANCE Ultra TR-FRET Detection Kit, 500 Assay Points to perform Cell Culture Contamination Detection Kit Bacteria

Get tips on using Prestained Protien ladder to perform Protein Ladder Prestained

Get tips on using pET-32c to perform Protein Expression Prokaryotic cells - E. coli rpf-like protein

Get tips on using EMBacY#5 to perform Protein Expression Eukaryotic cells - Hi5 Soluble G protein (Hendra Virus)

Get tips on using pTip-QC2-gi_21218674 to perform Protein Expression Prokaryotic cells - R. erythropolis putative DNA-binding protein

Get tips on using pET30a(+)-karp to perform Protein Expression Prokaryotic cells - E. coli 56‐kDa O. tsutsugamushi strain Karp protein

Get tips on using pTip-QC2-gi_21221796 to perform Protein Expression Prokaryotic cells - R. erythropolis putative tetR-family transcriptional regulatory protein

Get tips on using ExpiCHO™ Expression System Kit to perform Protein expression and purification Mammalian cells - CHO-K1 SUMO protein

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.