Protein expression and purification Mammalian cells HEK 293

Get tips on using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb #3398 to perform Autophagy assay cell type - HEK 293

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - HepG2

Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - HEK293T

Protein ladders are a set of standards known as molecular weight proteins that are utilized to identify the approximate size of a protein molecule run on a PAGE gel electrophoresis. The challenges in running the ladders are the choice of appropriate protein standard as it is used as visual evidence of protein migration, transfer efficiency, and positive control. Suitable protein markers can be selected on the basis of required properties and applications, i.e., fluorescent ladder, IEF, 2D SDS-PAGE ladder, natural ladder with an isoelectric point, and optimized ladders for Western Blot chemiluminescence detection. The key factors for running a distinct protein ladder are buffer conditions, charge/voltage at migration time, and the gel's concentration.

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Get tips on using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) to perform Cell cytotoxicity / Proliferation assay cell type - HEK 293

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - MLS-1765

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - BHK-21

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.