Protein expression and purification Insect cells

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 pMXs-IRES-Bsd Retroviral Expression Vector to perform CRISPR Human - Repression DDX3Y

Get tips on using pMXs-IRES-Bsd Retroviral Expression Vector to perform CRISPR Human - Repression DDX3X

Get tips on using Qubit™ Protein Assay Kit to perform Protein quantification Mammalian cells - OUMS-27

Get tips on using NanoOrange™ Protein Quantitation Kit to perform Protein quantification Mammalian cells - Human podocytes

Get tips on using CytoBuster™ Protein Extraction Reagent to perform Protein isolation Mammalian cells - CHO-K1

Get tips on using pgMAX system-rabbit voltage-dependent calcium channel β2a subunit to perform Protein Expression Prokaryotic cells - E. coli rabbit voltage-dependent calcium channel β2a subunit

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Mammalian cells - 6B5N

Get tips on using Pierce™ BCA Protein Assay Kit to perform Protein quantification Mammalian cells - A7r5

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Mammalian cells - A7r5