Protein Expression Eukaryotic cells T. thermophila

Get tips on using pwPICZalpha-DT390-bi-pIL-2-Gly to perform Protein Expression Eukaryotic cells - P. pastoris Porcine IL-2 fusion toxins

Get tips on using pFastBac-GP67-H6HA1-His-RhPV-IRES-EGFP to perform Protein Expression Eukaryotic cells - S. frugiperda HA1 of H6N1 AIV

Get tips on using ExpiCHO™ Expression System Kit to perform Protein expression and purification Mammalian cells - CHO-K1 SUMO protein

Get tips on using pwPICZalpha-DT390-bi-pIL-2-Non-N-Gly to perform Protein Expression Eukaryotic cells - P. pastoris Porcine IL-2 fusion toxins

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.

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 Purified Mouse Anti-SV40 Large T Antigen Clone PAb 101 (RUO) to perform Immunohistochemistry Mouse - SV40