Adapted from Leung et al (2008) Traffic 9:1698
For the separation of soluble and membrane fractions, enabling the initial characterisation of membrane-associated proteins.
- Phosphate Buffered Saline (PBS)
- Hypotonic Lysis Buffer (10 mM Tris-HCl, pH 7.5)
- Sample Lysis Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40)
- 2x Sample Buffer (124 mM Tris-Cl, pH6.8, 4.6% SDS, 10% β-mercaptoethanol, 20% glycerol, bromophenol blue)
NB: Lysis and sample buffers should contain protease inhibitors – this precludes the need to boil the samples (boiling can interfere with SDS-PAGE fractionation of membrane-associated proteins and should, therefore, be avoided)
- Wash 1×108 cells two times in PBS (1000g 10 minutes [~25ml], then 4500g 1 minute [~1ml] in a microfuge)
- Resuspend in 100 µl of COLD hypotonic lysis buffer and incubate on ice 5 minutes
- Centrifuge for 10 minutes in a microfuge at top speed, 4C (cold room)
- Transfer supernatant to a new eppendorf and add an equal volume of 2x sample buffer (soluble fraction)
- Resuspend pellet with 100 µl cold hypotonic lysis buffer and centrifuge as above
- Transfer supernatant to a new eppendorf and add an equal volume of 2x sample buffer (wash)
- Resuspend pellet in 100 µl of ice cold 1x sample lysis buffer and incubate on ice 25 minutes to lyse membranes. Add 2x sample buffer (membrane fraction)
Store all samples at -20°C.
Fractionate samples by SDS-PAGE (prior to loading vortex for ~5 sec to reduce the viscosity due to the presence of DNA).