The study of membrane-bound proteins, like trans-membrane receptors, can be difficult due to their relative low abundance in total cell lysates and their hydrophobic nature. I came across the ProteoExtract® Native M-PEK Kit when I was starting up my project in EGFR investigation. The kit provides all the reagents required for the extraction, including the Wash Buffer, 2 Extraction Buffers (I & II) and Protease Inhibitor Cocktail.
Usually, I would first seed cells according to the manual’s suggestion, i.e. 5 x 106 adherent cells in a T25 flask. After all the drug treatments, the culture medium was discarded. Then the cell monolayer was washed twice with 2 x 2 ml pre-chilled Wash Buffer. After aspirating the Wash Buffer completely, I mixed 2 ml ice cold Extraction Buffer I with 10 ul of the Protease Inhibitor Cocktail (and I also added 20 ul of a phosphatase inhibitor cocktail from Sigma) and spread the buffer to cover the cell layer. The flask was then incubated at 4ºC for 10 min; I put the flasks in a 4ºC refrigerator and rocked them gently at 2 min intervals. The resulting fraction is enriched in soluble proteins. After complete removal of this fraction, 1 ml ice cold Extraction Buffer II was mixed with 5 ul Protease Inhibitor Cocktail (and 10 ul phosphatase inhibitor cocktail from Sigma) and added to cover the cell layer. This time the flask was incubated at 4ºC for 30min, with occasional rocking. This second fraction is enriched with membrane proteins.
If you quantify your fractions with protein assays like BioRad’s DC Assay Kit, you will have extremely low protein concentrations. Don’t panic, your protein is there, but you may need to adjust your ‘original concepts’ on protein amounts that should be loaded on a SDS PAGE. For total cell lysates, I usually loaded 15 ug proteins per lane. However, 4 ug of the enriched fraction per lane can be just fine.
Figure 1: Upper panel - membrane fraction, Lower panel - soluble fraction
The downside of this kit is that the volume of the sample is large resulting in relatively low protein concentration. Every single sample resulted in 2 ml of soluble fraction and 1 ml of membrane fraction. In contrast, I usually lysed a cell pellet from a T25 flask with 100-200 ul lysis buffer. If you are testing a series of drug concentrations, or a panel of different drugs, or treating cells under different conditions, and at the same time, you don’t want to freeze and thaw your samples too much so you decide to aliquot your samples, you better buy a bigger freezer in your lab.
For your reference, I have tried to use 1.5 ml Extraction Buffer I and 0.8 ml Extraction buffer II instead, and the result is OK.
Graduate Student
Department of Pathology
The University of Hong Kong