The integrity of soluble and membrane proteins isolated was verified by SDS-PAGE. Further, the purity of membrane protein was confirmed by Western blot using an anti-sodium potassium ATPase antibody, which is a plasma membrane marker protein [39 (link)]. Proteins were separated by SDS-PAGE on 12% acrylamide gels prior to transfer onto the nitrocellulose membrane. Blot was developed using the Membrane Fraction WB Cocktail (Cat No. ab140365abcam), which contains 5 monoclonal Abs each targeting proteins located in different compartments of the cell. After primary and secondary antibodies’ incubations, the blot was developed using the ECL kit (Amersham GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol. The presence of the plasma membrane protein is shown by the anti-sodium potassium ATPase corresponding band (~112 kDa).
Membrane fraction wb cocktail
Manufactured by Abcam
Sourced in United Kingdom
Membrane Fraction WB Cocktail is a reagent designed for the preparation of membrane protein fractions from cell and tissue samples. It is suitable for use in western blotting applications.
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Lab products found in correlation
2 protocols using membrane fraction wb cocktail
1
Verification of Protein Isolation and Purity
2
Subcellular Fractionation and Identification
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Cells were mildly homogenized in ice-cold isotonic medium (40 million/mL, 250 mM sucrose, 20 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl 2 ) pH 7.6 supplemented with protease inhibitors in a 2 mL Dounce-tissue grinder (SigmaAldrich, Mexico) for 5 min. Nuclei were removed by centrifugation at 250 g, 4 °C for 10min, the supernatant was applied on top of a 4 mL of 27.4% Percoll (Pharmacia, Sweden). Ultracentrifugation was performed at 65 000 g, 4 °C for 60 min (MTX 150 Sorvall, rotor S560-ST, ThermoScientific). 50 μL fractions were collected and individual subcellular compartments were identified using the Membrane Fraction WB Cocktail (Abcam, UK). For IEF, the samples were diluted 1:4 using the sample/rehydration buffer.
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