Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.
The complete cell lysis reagent is a nondenaturing detergent formulation that dissolves cell membranes and extracts total soluble cellular protein in only 5 minutes. M-PER Reagent requires little or no mechanical disruption and yields more protein than freeze/thaw cycles and sonication. This mammalian cell lysis reagent is so efficient that adherent cells do not need to be scraped from the culture dish, enabling easy and direct lysis and analysis of cells grown in 24-well and 96-well plates. Resulting cell lysates from either adherent and suspension cells are compatible with many downstream assays including immunoassays, enzyme assays and a variety of common reporter assays.
- Gentle – mild detergent lysis, yielding extracts that are immediately compatible with coomassie (Bradford) and BCA protein assays or SDS-PAGE
- Compatible – extracts soluble proteins in nondenatured state, enabling direct use in immunoprecipitation and other affinity purification procedures
- Amine-free and dialyzable – formulation ensures compatibility with subsequent assay systems
- Convenient – lyse adherent cells directly in plate or after scraping and washing in suspension
- Non-denaturing – maintain activity of luciferase, beta-galactosidase, CAT and other reporter genes as well or better than other suppliers' products and freeze/thaw methods
Protein extraction is usually the first key step in any proteomics analysis procedure, and the cells must first be lysed to open the cell and release the proteins of interest. Several methods are commonly used to physically lyse cells, including mechanical disruption, liquid homogenization, sonication, freeze/thaw cycles and manual grinding.
Comparison of M-PER Reagent with freeze-thaw cycles, sonication and Brand P lysis buffer. COS-7 cells grown in 100mm plates at full confluency were washed once with 10mL of PBS, scraped with 1mL of PBS and centrifuged at 5000 rpm for 5 minutes to collect the cells. The cell pellets were resuspended in 0.5mL of respective extraction reagents and subjected to total protein extraction. For freeze-thaw cycles, the cell suspension in PBS was frozen in a dry ice and isopropanol bath for 10 minutes and thawed in a 37°C water bath. The freeze/thaw cycle was repeated three times. For sonication, the cell suspension was sonicated for 2 minutes with a 50% pulse using a Branson Sonifier* 450 Sonicator. For extraction with M-PER Reagent and Brand P lysis buffer, the cell suspensions were shaken for 5 minutes.The cell debris was removed by centrifugation at 13,000 rpm for 5 minutes and the supernatants were assayed for protein concentration by the BCA method.
|M-PER Reagent compatibility with reporter assays in transiently transfected mammalian cells. FM2 cells were transiently transfected with a reporter construct containing the luciferase gene. The transfected cells were lysed with either M-PER Reagent or Brand P lysis buffer and subjected to luciferase assay. For β-galactosidase and CAT assays, MDA-MB-231 cells were contransfected with reporter constructs expressing β-Gal and CAT, respectively. The transfected cells were lysed with M-PER Reagent or the freeze-thaw method, and the lysates were assayed for activity.
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