CelLytic™ MEM Protein Extraction Kit
SIGMA/CE0050
Synonym: Membrane protein extraction kit
Product Type: Chemical
Cells are coated with CTB-HRP (C3741), which binds to the membrane raft-residing monoganglioside GM1. Cells are then lysed on ice in Lysis and Separation buffer (L2417), the lysate is cleared, warmed to 30°C, and the phases are separated by centrifugation. The hydrophobic phase can be washed with Wash buffer (W2514) to remove residual hydrophillic proteins from the hydrophobic phase. Proteins extracted into the hydrophobic and hydrophilic phases can be analyzed by Western blot. Proteins can be analyzed by SDS-PAGE followed by Western blot (using non-coated cells). Lysis and Separation buffer contains 150 mM Sodium chloride. Some proteins may require a higher salt concentration for effective extraction and separation. The alternative assay tests the extraction of connexin-43, a membrane hydrophobic protein and caveolin-1, a raft associated 20 kDa protein, into the hydrophobic phase, and MAP kinase, cytosolic protein, into the hydrophilic phase. The alternative assay requires running of 2 gels followed by Western blots. One blot is developed with both anti connexin–43 and anti caveolin-1 antibodies. The second gel is developed with anti MAP kinase antibody.
| Quality Level | 200  |
| shipped in | wet ice |
| storage temp. | −20°C |
| technique(s) | protein extraction: suitable |
| Application: | CelLytic™ MEM Protein Extraction Kit has been used for de novo lipogenesis measurements using hepatocytes and to extract membrane proteins for western blotting. |
| Application: | Sufficient reagents supplied for 80 tests. |
| General description: | CelLytic™ MEM Protein Extraction Kit offers a fast and convenient method to isolate hydrophobic and raft microdomain associated proteins from cells. The method is based on phase separation and does not require cell membrane isolation. The separated proteins can be used for further experiments such as SDS-PAGE, Western blotting, dot blotting, and immunoprecipitation. The kit has been tested on, but not limited to, HeLa, HEK-293, NIH 3T3, COS and CHO cell lines. |
| General description: | Membrane proteins make up around 20-30% of an organism′s genome and serve as cellular gatekeepers, regulators, and sensors. They have diverse cellular functions, such as shielding the cell from external toxins, being the starting point of intracellular signaling cascades, and retaining critical ion concentrations. |
| Legal Information: | CelLytic is a trademark of Sigma-Aldrich Co. LLC |
| Flash Point(F) | 188.6 °F - closed cup |
| Flash Point(C) | 87 °C - closed cup |
| Storage Temp. | −20°C |
| UNSPSC | 41116134 |
| Components | Lysis and Separation Buffer 50 mL; Wash Buffer for CelLytic MEM 50 mL; Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution 1 mL; Sodium Chloride, 4M Solution 1.5 mL |