This experiment describes the Neville method for isolation of liver plasma membrane. This experiment is from the guide to protein purification and characterization by Houzhu Zhu.
Operation method
Neville's method for the separation of liver plasma membrane
Materials and Instruments
Chopped rat liver Move MATERIALS AND EQUIPMENT For more product details, please visit Aladdin Scientific website.
Solution A Sucrose Solution B
Doimce glass homogenizer Centrifuge tubes Refractometer SW-28 Turning head and thin-walled polyallomer SW-28 tubes
Chopped rat liver (~25 g; cryopreserved until use)
NOTE: Human placenta is an excellent raw material for the production of insulin receptors. Unfortunately, human placenta may be difficult to obtain for researchers not affiliated with the Medical Center.
Doimce's glass homogenizer (15-ml) with pestle and mortar A and B
Coarse cloth (cheese wrap)
Centrifuge tubes (250-ml)
Refractometer (No. 13_947; Fisher Scientific or other brands)
SW-28 Turning head and thin-walled Polyallomer SW-28 tubes
Reagents
Solution A
Sucrose [69% (w/w) and 42.3% (w/w)]
Solution B
(for formulae, see "Preparation of Reagents" PP.234-240)
Procedure
This procedure is a modification of the method of Neville (1968). All operations are carried out at 4°C.
1) Take about 68 chopped mouse livers and place them in a medium-sized (15-ml) Dounce's glass homogenizer. Add 8 ml of ice-cold Solution A, place in an ice bath and pestle and mortar with a loose B pestle 8 times. Work slowly so as not to spill the homogenate. Then pour the homogenate into 500 ml of Solution A (in an ice bath). Repeat this procedure for the remaining 18 g of livers in three batches and pour the homogenate into the 500 ml of Solution A mentioned above. The final mixture is stirred for 3 min, filtered through a double layer of coarse cloth and collected in a 2000-ml flask.
Note: When preparing tissue homogenates, the pestle and mortar should be pulled up and down to create a shearing force; twisting is not helpful. If it is too difficult to pull the pestle, remove it slowly and wipe away any connective tissue tangles attached to the ball of the pestle with a piece of paper. Do not press down on an unsupported homogenizer as this may break it. Hold the homogenizer firmly in your hand while supporting it in the ice cylinder. Avoid air bubbles in the homogenate.
2) Divide the filtered homogenate into 6 250-m centrifuge tubes and centrifuge at 1500 g (JA-14 turn head, 3200 r/min) for 10 min at 4°C. Pipette the supernatant slowly but do not disturb the loose sediment. Then transfer the precipitate to a 15-ml Doimce's homogenizer placed in an ice bath.
3) Homogenize the precipitate with three gentle swipes of a loose pestle and mortar B. Measure the volume of the homogenate.
4) Pour the homogenate into a beaker. Add an equal volume of 69% (w/w) sucrose solution and stir to mix until no visible flocs remain. Check the sucrose concentration of the solution with a refractometer. Adjust with water or 69% sucrose solution so that the sucrose-homogenate mixture contains 44% sucrose (refractive index 1.4076).
Note: When taking refractometer measurements, mix the homogenate well between each reading.
5) Pour the homogenate into a number of SW-28 centrifuge tubes, 20 ml per tube.
6) Carefully top off each portion of homogenate with 10 ml of 42,3% (w/w) chilled sucrose solution (refractive index 1.4042).
7) Equilibrate the centrifuge tubes with the 42,3% chilled sucrose solution.
8) Place the tubes in a cold SW-28 rotor at 25,000 rpm. Centrifuge the tube in a cold SW-28 rotor head at 25,000 r/min (90,000 g) for 2 h. Check the vacuum grease on the 0-ring to make sure the rotor head is not leaking.
9) Using a spatula, remove the 2-4 mm thick membrane layer floating on the top of the centrifuge tube and transfer it to the same SW-28 tube. Add Solution B and mix gently. Centrifuge at 25000r/min for 30 min at 4°C to collect the membrane fraction, this step removes sucrose. Discard the supernatant and add 3 ml of Solution B to the precipitate. The precipitate is gently homogenized in a Dourtce's homogenizer (15-ml) with a pestle and mortar, and aliquots of the suspension are dispensed into microcentrifuge tubes at 0.25 ml each and frozen at -20°C. The precipitates are then frozen at -20°C.