Protein expression and purification Insect cells Hi5

Get tips on using High Pure RNA Isolation Kit to perform RNA isolation / purification Cells - immortalized CMT12

Get tips on using High Pure RNA Isolation Kit to perform RNA isolation / purification Cells - immortalized A2780

Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Tissue - Human aortic endothelial 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.

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

Get tips on using Direct-zol RNA Kits to perform RNA isolation / purification Cells - primary human renal proximal tubular epithelial cells

Get tips on using RNeasy Lipid Tissue Mini Kit to perform RNA isolation / purification Cells - primary human brain microvascular endothelial cells

Get tips on using PureLink™ RNA Mini Kit to perform RNA isolation / purification Cells - primary human umbilical vein endothelial cells

Get tips on using ISOLATE II RNA Micro Kit to perform RNA isolation / purification Cells - primary human carotid artery endothelial cells

Get tips on using MagNA Pure Compact RNA Isolation Kit to perform RNA isolation / purification Cells - primary porcine tracheal epithelial cells