Protein isolation Mammalian cells - HOG

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 3 matching solutions for this experiment

RIPA Lysis Buffer System

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	- Cells are thawed on ice and resuspended in 3-5X packed-cell volume of ice-cold RIPA Lysis Buffer System according to the manufacturer’s instructions and freshly supplemented with 1X PhosSTOP phosphatase inhibitor cocktail when applicable.	- Cell pellets are homogenized by 5-10 passages through a 21-gauge needle then mixed for 60 minutes at 4 °C on an automatic rotator. Insoluble cellular debris is pelleted using centrifugation at 13000 rpm for 10 mins at 4 °C.

Protocol tips
- Cells are thawed on ice and resuspended in 3-5X packed-cell volume of ice-cold RIPA Lysis Buffer System according to the manufacturer’s instructions and freshly supplemented with 1X PhosSTOP phosphatase inhibitor cocktail when applicable.
Downstream tips
- Cell pellets are homogenized by 5-10 passages through a 21-gauge needle then mixed for 60 minutes at 4 °C on an automatic rotator. Insoluble cellular debris is pelleted using centrifugation at 13000 rpm for 10 mins at 4 °C.

Manufacturer protocol Publication protocol 1 Relevant paper

Nuclear Extract Kit

Active Motif

Upstream tips	Protocol tips	Downstream tips
- Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 mM NaF, 2 mM imidazole, and a cocktail of protease inhibitors.	- Nuclear Extract kit was used accordingly to the manufacturer’s instructions.

Upstream tips
- Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 mM NaF, 2 mM imidazole, and a cocktail of protease inhibitors.
Protocol tips
- Nuclear Extract kit was used accordingly to the manufacturer’s instructions.

Manufacturer protocol Publication protocol 1 Relevant paper

Subcellular Protein Fractionation Kit for Cultured Cells

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	- Instructions are followed regarding the manufacturer protocol, except fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer

Protocol tips
- Instructions are followed regarding the manufacturer protocol, except fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer

Manufacturer protocol Publication protocol 1 Relevant paper

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