Pharmacological agents may be added to a Membrane Mix made in step 2 of the Basic Protocol to test the involvement of Golgi coat proteins and MT motor proteins in organelle movement in vitro.  For review of the effects of these pharmacological agents on membrane trafficking, see Klausner et al., 1992.  These should be added to the mix, correcting all components for concentration, and incubated at 37oC for 15 minutes prior to introduction into the flow chamber.

Equipment:  TLA tabletop ultracentrifuge (Beckman) equipped with TLS-55 rotor and  ultraclear tubes (Beckman # P5-1102).

 The following are some suggestions for agents to add to the Membrane Mix
 -60 mm Brefeldin-A to remove Golgi coat proteins
dilute a 1.5 ml of a 3.6 mM ethanolic stock of Brefeldin A 1:1 into PEM just prior to use, then add 3 ml to the Membrane Mix
 -Aluminum Fluoride-  to activate heterotrimeric G- proteins
  add 1 ml of 30 x NaF and 1ml of 30 x AlCl3 to the membrane Mix
-1 mM MgGTP-g-S- to activate heterotrimeric G- proteins
add 1 ml 30mM MgGTP-g-S to the Membrane Mix
-5 mM MgAMP-PNP to inhibit kinesin-like proteins (Vale et al., 1985)
add 1 ml of 150 mM Mg AMP-PNP to the Membrane Mix
 -25uM Sodium Orthovanadate to inhibit cytoplasmic dynein (Shpetner et al., 1988)
Dilute 1 ml of 100 mM stock into 133 ml of PM buffer
Add 1 ml of this dilution to the Membrane Mix

You may also pre-treat the organelles to strip off subsets of peripheral proteins prior to adding them to the mix.  This tests the involvement of these proteins in organelle movement.  Treatments include:
 -0.6 M KI ("salt-washed organelles")  (add from a 3 M KI stock)
 -10 mM EDTA ("EDTA-stripped organelles") (add from 0.5 M EDTA stock)
 -150 mM Na2CO3 ("carbonate-washed organelles")  (add from a 0.5 M Na2CO3 stock, pH=11.5)

To strip membranes with any of these three agents
1)  Incubate a small aliquot (~50-100 ul) of isolated organelles with the chosen agent at the noted concentration for 30 min on ice.
2)  Transfer to a mini-ultracentrifuge tube (Beckman # P5-1102) and pellet the membranes in a Beckman TLA tabletop ultracentrifuge for 1 hour at 50,000 RPM in the TLS-55 s(Beckman) swinging bucket rotor at 4oC.
3) Carefully remove the supernatant and resuspend the membrane pellet by gentle trituration in the original volume of PEM containing 250 mM sucrose.

Another idea is to incubate membranes, cytosol, and energy mix with brefeldin-A, aluminum fluoride or GTP-g-S (1/2 hour at 37oC) prior to adding them to the mix, then isolate these membranes by centrifugation as above, resuspend them and use them as per stripped membranes.

*=As published in...
Waterman-Storer, C.M.  (In press)  Microtubule/organelle motility assays.  In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds.  John Wiley, NY.


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