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

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.

Get tips on using MethylFlash™ Methylated DNA Quantification Kit to perform DNA methylation profiling Whole genome profiling - MCF-7, MDA-MB-453 human breast cancer

Get tips on using CM-H2DCFDA (General Oxidative Stress Indicator) to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Get tips on using Carboxy-H2DCFDA (general oxidative stress indicator) to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Get tips on using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit to perform Cell line authentication Human iPSC cells derived from peripheral blood mononuclear cells

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - MCF-7 human breast cancer cells

Get tips on using Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells to perform Live / Dead assay mammalian cells - FE002-SK2 human skin progenitor cells

Get tips on using FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme to perform TUNEL assay cell type - A127, U87MG, U251MG, T98G human glioblastoma cells

Get tips on using OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence) to perform ROS assay cell type - human umbelical vein endothelial cells (HUVEC)

Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.