Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation

Abstract

Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material.

Lysine 63 -linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth

“…For Western blotting and IP, 5-20 mg or 150-200 mg, respectively, of root material [3-6 d after germination (DAG)] was homogenized and processed as described in ref. 38. Drug and growth regulator treatments were performed as indicated.…”

A PP6-Type Phosphatase Holoenzyme Directly Regulates PIN Phosphorylation and Auxin Efflux in Arabidopsis

“…The membrane protein extraction was performed as previously described (Abas and Luschnig, 2010), except that the protein phosphatase inhibitors were excluded from the extraction buffer. The membrane fractions were eventually solubilized in 0.1% Brij35 and preheated at 65°C for 10 min to inactivate the endogenous enzymes.…”

“…The reactions were stopped by adding 23 sample buffer (4% SDS, 250 mM Tris-HCl, pH 6.8, 80 mM dithioerythritol, and 40% glycerol) and heated. Samples were separated as described (Abas and Luschnig, 2010) and probed with GFP antibodies (Invitrogen; 1:1000). The second antibody, goat anti-rabbit IgG peroxidase antibody (Sigma-Aldrich), was used at 1:10,000.…”

Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (<i>Oreochromis niloticus</i>)

“…Microsomes can feasibly be isolated when eukaryotic cells are dissolved and centrifuged (30). Many organs of fish have been used to extract microsomes.…”