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.
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.
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.
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.
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.
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.
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 miRNA Complete Labeling and Hyb Kit to perform Microarray RNA amplification & Labeling - Human endometrial stromal cells Cyanine 3-pCp
Get tips on using JetPrime to perform DNA transfection Mammalian cells - Immortalized cell lines COS7
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been greatly used for studies on embryonic development and cell differentiation.iPSCs provide a stable source for either self-renewal or differentiation into suitable cells when cultured in a particular environment. Pluripotent cell culture was originally started by deriving cells from inner cell mass (ICM) from pre-implanted blastocysts, these were called embryonic stem cells. These cells after isolation can be grown on traditional extracellular matrices (like mouse embryonic fibroblasts, MEFs) or feeder-free culture systems. DMEM/F12 has been the most commonly used basal media in the culture of pluripotent cells. These cells are cultured at normal atmospheric oxygen levels, 21%, however, some studies have proposed that 4% oxygen tension may be better for hESC growth. Higher D-glucose concentration (4.2g/l) and osmolarity (320mOsm) that mimics the natural environment of embryonic tissue are optimal for the growth of hESCs. Supplements like N2 and/or B-27, in the presence of growth factors like bFGF, have been shown to increase pluripotency of these cells. bFGF, FGF2 and other ligands of receptor tyrosine kinases like IGF are also required or maintain self-renewal ability of these cells. TGF𝛃1, by its activation of SMAD2/3 signalling, also represses differentiation of iPSCs. Other compounds like ROCK inhibitors reduce blebbing and apoptosis in these cells to maintain their clonogenicity. However, an inhibitor for LIF (leukaemia inhibitory factor, which is one of the pluripotent genes) has an opposing effect. Therefore, it is important to understand the culture conditions and media composition that affect downstream signalling in hESCs or iPSCs that may lead to their differentiation.
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