Site Directed Mutagenesis (SDM) Human Point mutation Caco-2

Get tips on using Anti-TIMP2 antibody [3A4] (ab1828) to perform Western blotting TIMP-2

Get tips on using Mouse BMP2 ELISA Kit (ab119582) to perform ELISA Mouse - BMP-2

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

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

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 pVRB2B3 to perform Protein Expression Eukaryotic cells - HEK293 hβ-defensin 2/3

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - immortalized C3H-10T1/2

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - immortalized U-2 OS

Get tips on using Fluoro hROS to perform ROS assay cell type - MiaPaCa-2 pancreatic carcinoma

Get tips on using RNeasy Mini Kit to perform RNA isolation / purification Cells - immortalized Saos-2