Site Directed Mutagenesis (SDM) Human Point mutation Caco-2

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - Caco-2

Get tips on using Monoclonal Anti-ATG5 antibody produced in mouse to perform Autophagy assay cell type - CaCo-2

Get tips on using Monoclonal Anti-ATG12 antibody produced in mouse to perform Autophagy assay cell type - CaCo-2

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of 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 VWR Life Science RIPA Lysis Buffer, Biotechnology Grade to perform Protein isolation Mammalian cells - Caco-2

Get tips on using LC3A/B (D3U4C) XP® Rabbit mAb #12741 to perform Autophagy assay cell type - CaCo-2

Get tips on using anti-p62 / SQSTM1 (C-terminus) guinea pig polyclonal, serum to perform Autophagy assay cell type - CaCo-2