Site Directed Mutagenesis (SDM) Human Point mutation THP-1

Get tips on using p-c-Jun Antibody (KM-1): sc-822 to perform Western blotting C-Jun

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 CD279 (PD-1) Monoclonal Antibody (RMP1-30), FITC, eBioscience™ to perform Flow cytometry Anti-bodies Mouse - CD279/PD-1

Get tips on using Laminin β-2 Antibody (H-1): sc-133241 to perform Western blotting Laminin subunit Beta-2

Get tips on using Laminin beta-2/gamma-1 Monoclonal Antibody (A5) to perform Western blotting Laminin subunit Beta-2

Get tips on using Quick Start™ Bradford Protein Assay Kit 1 to perform Protein quantification Mammalian cells - BV-2

Get tips on using anti-alpha-Smooth Muscle Actin mouse monoclonal, ASM-1 to perform Immunohistochemistry Alpha smooth muscle Actin - Mouse -NA- -NA-

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.