RNA isolation / purification Cells Cancer cell lines

Get tips on using Iscove's Modified Dulbecco's Medium (IMDM) (ATCC® 30-2005™) to perform Mammalian cell culture media KG1

Get tips on using DMEM High Glucose w/ L-Glutamine w/o Sodium Pyruvate to perform Mammalian cell culture media U87

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

Get tips on using Galacto-Light Plus™ β-Galactosidase Reporter Gene Assay System to perform Reporter gene assay β-galactosidase substrates - U87 and U251 glioblastoma cells

Get tips on using LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13082 to perform Autophagy assay cell type - RAW 264.7

Get tips on using ViralSEQ™ Quantitative Sf-Rhabdovirus Kit with PrepSEQ™ Nucleic Acid Sample Prep Kit to perform Cell Culture Contamination Detection Kit Virus

Get tips on using OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence) to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.