Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - Human Neuroblastoma cell lines_SK-N-BE(2)-C
Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.
Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - Human Neuroblastoma cell lines_SK-N-BE(2)-C
Get tips on using Lectin from Triticum vulgaris (wheat) to perform Western blot 1,4 β-N-acetyl-D-glucosamine - Triticum vulgaris Bacteria Biotin
Get tips on using Anti-NAPSIN A antibody (ab187300) to perform Immunohistochemistry Human - Naspsin A
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.
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Get tips on using pET32(a)+-BLS to perform Protein Expression Prokaryotic cells - E. coli BLS
Get tips on using M-PER™ Mammalian Protein Extraction Reagent to perform Protein isolation Mammalian cells - Human Neuroblastoma cell lines_SK-N-BE(2)-C
When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.