I need to perform binding and purify a plasma protein that binds a tetrapeptide, using plasma membranes from adult rat cardiac fibroblasts. Could anyone suggest to us a reliable method for membrane fractionation so that we could work on only membranes void form any cytosolic contaminants?
If you'd like to simply purify all membranes, there's a very simple method:
1. lyse your cells in a hypotonic, detergent-free buffer (e.g. 50mM Tris + 5mM EDTA) and homogenize with a Dounce or a 27-gauge needle
2. pellet nuclei at 300xg for 5min
3. then a centrifugation at 100,000xg for 30mins should pellet (P100) nearly all membranes while leaving cytoplasmic material in the supernatant (S100)
A really nice method for more specifically isolating plasma membranes for biochemical and biophysical studies is described here:
Nature Protocols 7 p1042 - p1051 03/05/2012
doi:10.1038/nprot.2012.059
http://www.nature.com/nprot/journal/v7/n6/full/nprot.2012.059.html
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