Protein Expression Eukaryotic cells K. lactis

Get tips on using pET-21a(+) DNA to perform Protein expression and purification Bacteria - Escherichia coli Prefoldin (PFD)

Get tips on using pQE-80L vector to perform Protein expression and purification Bacteria - Escherichia coli IFABP-Aβ

Get tips on using pPIC9K Pichia Vector to perform Protein expression and purification Yeast - Pichia pastoris N-APP

Get tips on using pGEX-6P-1 Vector to perform Protein expression and purification Bacteria - Escherichia coli FleN

Get tips on using pQE-30 Xa Vector to perform Protein expression and purification Bacteria - Escherichia coli Siva1

Get tips on using Micro BCA™ Protein Assay Kit to perform Protein quantification Colorimetric method

Get tips on using Pierce™ BCA Protein Assay Kit to perform Protein quantification Colorimetric method

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 has 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 the efficacy of transduction and shRNA on its target site.

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Tissue - mouse kidney

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Tissue - mouse liver