Protein expression and purification Mammalian cells HeLa

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 jetPEI® DNA transfection, HTS application to perform DNA transfection Mammalian cells - Immortalized cell lines Chang Liver cells

Get tips on using jetPEI® DNA transfection, HTS application to perform DNA transfection Mammalian cells - Primary cells Rat dermal fibroblasts (rDF)

Get tips on using Zombie Aqua™ Fixable Viability Kit to perform Live / Dead assay mammalian cells - human peripheral blood mononuclear cells

Get tips on using Maxwell® 16 LEV simplyRNA Purification Kit to perform RNA isolation / purification Tissue - Human Muscles

ROS has a very short half-lives in biological environment as they are influenced by exposure to ambient oxygen. As it is highly reactive and hard to measure care should be taken to ensure the stability of the sample during isolation, preparation, storage, and analysis.

Get tips on using Wizard® Plus Midipreps DNA Purification System Technical Bulletin to perform Plasmid Isolation Proteus mirabilis

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform siRNA / RNAi /miRNA transfection Human Cells - HT-1376 ROCK2

Get tips on using N788 phenol-free total RNA purification kit to perform RNA isolation / purification Bacteria - Gram negative Pseudomonas aeruginosa