siRNA / miRNA gene silencing Mouse mESC

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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 EZ-ChIP™ to perform ChIP Rat - Mesenteric arteries

Get tips on using Autophagy Detection Kit to perform Autophagy assay cell type - Mesenchymal stromal cells

Get tips on using AquaRNA Kit to perform RNA isolation / purification Cells - primary human mesenchymal stem cells

Get tips on using RNeasy Mini Kit to perform RNA isolation / purification Cells - primary human mammary mesenchymal cells

Get tips on using StemSpan™ SFEM to perform Stem cell Differentiation media hiPSCs differentiation into mesodermal lineage cells

Get tips on using Minimum Essential Medium Eagle to perform Stem cell culture media Human bone mesenchymal stem cell (BMSC)

Get tips on using ChIP-IT® Express Chromatin Immunoprecipitation Kits to perform ChIP Rat - Mesenteric arteries

Get tips on using TRI Reagent® to perform Protein isolation Mammalian cells - Mouse_Brown fat