dna-isolation-purification-cells-primary-cells-mouse-embryonic-fibroblast-mef

Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Cells - primary mouse dorsal root ganglion neurons

Get tips on using miRNeasy Mini kit to perform RNA isolation / purification Cells - primary mouse dorsal root ganglion neurons

Get tips on using PureLink™ RNA Mini Kit to perform RNA isolation / purification Cells - primary mouse cortical neurons

Get tips on using Anti-SQSTM1 / p62 antibody (ab56416) to perform Autophagy assay cell type - MEFs (mouse embryonic fibroblasts)

Get tips on using Anti-p62 (SQSTM1) (Human) pAb to perform Autophagy assay cell type - MEFs (mouse embryonic fibroblasts)

Get tips on using MagNA Pure Compact RNA Isolation Kit to perform RNA isolation / purification Cells - primary human mesenchymal stem cells

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.