DNA transfection Mammalian cells Primary cells

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.

Get tips on using SV Total RNA Isolation System to perform RNA isolation / purification Cells - primary human coronary artery smooth muscle cells

Get tips on using PureLink™ RNA Mini Kit to perform RNA isolation / purification Cells - primary canine coronary artery smooth muscle cells

Get tips on using E.Z.N.A.® Total RNA Kit I to perform RNA isolation / purification Cells - primary bovine umbilical vein endothelial cells

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human airway epithelial cells

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human aortic endothelial cells

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 VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal Primary Antibody to perform Immunohistochemistry Human - MSH2

Get tips on using Cell DNA Isolation Kit to perform DNA isolation / purification Cells - Immortalized cell lines C2C12

Get tips on using QIAamp DNA Mini Kit to perform DNA isolation / purification Cells - Immortalized cell lines 3T3