Protein isolation Mammalian cells - BHK21

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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Found 4 matching solutions for this experiment

CelLytic™ M

Sigma-Aldrich

Protocol tips
Cells growing in log phase were transfected with the indicated RNA at 50–75% confluence using TransIT-mRNA transfection kits (Mirus Bio, Madison, WI, USA). After 72 h, the cells were lysed, and proteins were extracted in CelLytic™ M Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich, St. Louis, Missouri) supplemented freshly with 25 U/μL benzonase (EMD Millipore, Billerica, MA, USA), and cOmplete, Mini EDTA-free Protease Inhibitors (Roche Life Science, Mannheim, Germany) according to the manufacturer’s recommendations.
NP40 Cell Lysis Buffer

Thermo Fisher Scientific

Protocol tips
The transfected cells were washed with phosphate-buffered saline and lysed in NP40 lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium duodecyl sulphate) containing a complete protease inhibitor cocktail (Boehringer-Mannheim, Mannheim). The cell extracts were centrifuged at full speed in a refrigerated Eppendorff centrifuge for 10 minutes.
Protocol tips

The nucleus and cytoplasm of MHV-68-infected BHK-21 cells were isolated using a CelLytic NuCLEAR extraction kit (product code NXTRACT; Sigma-Aldrich) according to the instructions in the manual accompanying the kit, with some modifications.
RIPA Lysis and Extraction Buffer

Thermo Fisher Scientific

Protocol tips
The cells were rinsed with chilled PBS three times and subject to RIPA lysis (Thermo). To purify surface protein, the lysates were mixed with NeutrAvidin-agarose beads (Thermo) at 4°C overnight.
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