protein-expression-and-purification-mammalian-cells-hek-293-mt-pa

Get tips on using TRIzol™ Plus RNA Purification Kit to perform RNA isolation / purification Cells - primary rabbit aortic endothelial cells

Get tips on using Wizard® Genomic DNA Purification Kit to perform DNA isolation / purification Cells - Primary cells Rat cortical neurons

Get tips on using Wizard® Genomic DNA Purification Kit to perform DNA isolation / purification Cells - Primary cells Human primary keratinocytes

Get tips on using Qproteome Bacterial Protein Prep Kit to perform Protein isolation Bacteria - Salmonella paratyphi A

Get tips on using Wizard® Genomic DNA Purification Kit to perform DNA isolation / purification Cells - Primary cells Mouse embryonic fibroblast (MEF)

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.

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

Get tips on using Biotinylated Protein Ladder Detection Pack #7727 to perform Protein Ladder Immunofluorescence

Get tips on using PageRuler™ Prestained NIR Protein Ladder to perform Protein Ladder Prestained

In ChIP, the most vital step is the binding of an antibody and choosing the right antibody. The binding affinity of different types of immunoglobulins to protein A or G differs significantly. Henceforth, it is recommended to choose either protein A or protein G coated beads. If you do not see any product in the positive control, add 5–10 μg of chromatin and 1–5 μg of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G/A beads. If no product is observed in the experimental sample, add more DNA to the PCR reaction or increase the number of amplification cycles. Furthermore, if you have any problem with antibodies, make sure to use the ChIP-validated antibody.