Protein isolation Mammalian cells - CHO-K1

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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RIPA Buffer

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Cells were lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P40, 2 mM EDTA, 50 mM NaF, 50 mM β-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were then passed through a 21-gauge needle followed by centrifugation at 4 °C for 30 min at 13,500g. The supernatant was used as a cell protein sample.

Protocol tips
Cells were lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P40, 2 mM EDTA, 50 mM NaF, 50 mM β-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were then passed through a 21-gauge needle followed by centrifugation at 4 °C for 30 min at 13,500g. The supernatant was used as a cell protein sample.

Protocol tips

Cells were lysed in RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P40, 2 mM EDTA, 50 mM NaF, 50 mM β-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were then passed through a 21-gauge needle followed by centrifugation at 4 °C for 30 min at 13,500g. The supernatant was used as a cell protein sample.

Manufacturer protocol Publication protocol 1 Relevant paper

CytoBuster™ Protein Extraction Reagent

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	The effectiveness of the successful procedure of cell lysis developed from this study was compared with the commercial lysis buffer CytoBuster Protein Extraction Reagent (Novagen, US). The procedure was as per manufacturer’s instructions as depicted in Figure 2.

Protocol tips
The effectiveness of the successful procedure of cell lysis developed from this study was compared with the commercial lysis buffer CytoBuster Protein Extraction Reagent (Novagen, US). The procedure was as per manufacturer’s instructions as depicted in Figure 2.

Manufacturer protocol Publication protocol 1 Relevant paper

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