Prepare sample for immunoadsorption

Example: Prepare sample enriched in stacked Golgi complexes from rat livers:

4. On the same day the immunoadsorption experiment is to be done, sacrifice a rat that has been starved overnight (to deplete hepatic glycogen) and remove liver. Weigh liver, mince, homogenize, and filter as for velocity differential centrifugation (see Basic Protocol 1), except homogenize at 20% (w/v) in 0.5 M sucrose/0.1 M potassium phosphate/5 mM MgCl2 homogenization medium.

This method for preparing a partially purifiedfraction enriched in stacked Golgi complexes from rat livers is based on the procedure ofLeelavathi et al. (1970).

5. Prepare a postnuclear supernatant (PNS) by centrifuging sample as for differential centrifugation by velocity (see Basic Protocol 1, step 4).

6. Place a4-ml cushion of 1.3 M sucrose/0.1 M potassium phosphate/5 mMMgCl2 into a 10-ml centrifuge tube, and layer 6 ml PNS on top. Centrifuge 60 min at 105,000 x g (in Type 50 rotor), 4°C.

7. Collect the band at the surface of the 1.3 M sucrose layer and adjust to 1.1 M sucrose using homogenization medium.

8. Prepare a second set of discontinuous gradients containing 6 ml each of 1.4 M, 1.3 M, and 1.25 M sucrose in 0.5 M potassium phosphate/5 mM MgCl2. Layer 6 ml of the adjusted sample (step 7) over each gradient, overlay with homogenization medium to fill the tubes, and centrifuge 90 min at 80,000 x g (in SW28 rotor), 4°C.

9. Collect the Golgi fraction at the interface between the load and the 1.1 M sucrose layer. Adjust to 0.25 M sucrose and 5 mg/ml BSA. Place on ice.

The Golgi fraction is now ready to be used for immunoadsorption (which should be done on the same day). The fraction should be assayed for protein content and maintained on ice until use.

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