Mouse Liver Fractionation Suitable for 2D
Ref: “Isolation of Plasma Membrane, Golgi Apparatus, and Endoplasmic Reticulum Fractions from Single Homogenates of Mouse Liver” Edward M. Croze and D. James Morre. Journal of Cellular Physiology 119:46-57
Make Solutions:
Basic Buffer:
37.5 mM Tris Maleate pH 6.4 9.12 g/L
5 mM MgCl2 10 ml of 0.5 M stock
Add 1% Dextran to 400 ml 4 g
Make sucrose solutions with the buffer with the dextran added
Sucrose Solutions:
0.5 M 1.2 M 1.3 M 1.5 M 2.0 M
17.112g 20.53g 22.24g 25.67g 34.23g Sucrose
100 ml 50 ml 50 ml 50 ml 50 ml Buffer
1 mM Na2HCO3
10 mM Tris pH 7.5
Sucrose Gradients for Plasma Membrane Isolation:
Made in ddH20
- 10 g Sucrose in 25 ml
- 10.65 g Sucrose in 25 ml
- 11 g Sucrose in 25 ml
- 11.5 g Sucrose in 25 ml
- 12 g Sucrose in 25 ml
- 13.35 g Sucrose in 25 ml
- 20.25 g Sucrose in 25 ml
Liver Fractionation
-liver harvested from 3 males – Balb/c – 5 weeks old.
-liver weighed and minced
-suspend liver in 6 ml 0.5 M Sucrose solution
-homogenize liver for 30-45 seconds at 4000 rpm
-centrifuge at 5000g (6000 rpm, JA17) for 15 minutes at 4 C
-supernatent is used for ER isolation, the top 1/3 of pellet is used for golgi isolation, and the lower ½ of pellet is used for PM isolation.
ER ISOLATION
-supernatant from above is diluted 1:4 with 0.5 M sucrose and centrifuged at 8500g (7250 rpm JA17) for 5 minutes to remove mitochondria. Save pellet for isolation of mitochondria.
-resulting supernatant is top loaded onto a discontinuous sucrose gradient:
6 ml 2 M Sucrose : 8 ml 1.5 M Sucrose : 1.3 M Sucrose : supernatant
-centrifuged 90 000g (25 000 rpm, SW28) for 90 minutes
-ER collected at interfaces 1.3/1.5 and 1.5/2.0
-pooled ER is diluted 1:3 with 10 mM Tris, pH 7.5
-centrifuge 25 000 rpm for 45 minutes (SW28)
-resuspend pellet in 10 mM Tris, pH 7.5
-centrifuge 25 000 rpm for 45 minutes (SW28)
-resuspend pellet in 2 ml 10 mM Tris, pH 7.5 and aliquot 200 ul into 10 small (T100-Weiner Lab) size tubes. Remember to save 25 ul for protein assay.
-centrifuge 63 000 rpm for 30 minutes in the Weiner Centrifuge
-remove supernatant and freeze pellet in –80.
Golgi Isolation
-yellow/brown portion (upper 1/3) of pellet was resuspended in a small part of supernatant with a large bore pipet. Transfer to a new tube.
-pipet up and down 12 times. Layer over 2.7 ml of 1.2 M Sucrose in SW60 size swinging bucket tubes and centrifuge 100 000g (30 000 rpm) in SW60 rotor
-golgi collected from interface with care not to remove any of the 1.2 M Sucrose layer (contains ER fragments)
-collected golgi are diluted in ddH20 and concentrated by centrifugation at 5500g (6500 rpm, JA17) for 20 minutes
-resuspend in 10 mM Tris, pH 7.5 and centrifuged at 5500g (6500 rpm, JA17) for 20 minutes
-resuspend again in 10 mM Tris, pH 7.5 and centrifuged at 5500g (6500 rpm, JA17) for 20 minutes
-resuspend pellet in 1 ml 10 mM Tris, pH 7.5 and aliquot 200 ul into eppendorf tubes and centrifuge in cold centrifuge at 14 000 rpm for 25 minutes. Remove supernatant and freeze pellet. Remember to save 25 ul for protein assay.
Plasma Membrane Isolation
-the lower ½ of pellet is resuspended and homogenized in 2.5 ml 1 mM Na2HCO3 until homogenous (200 rpm)
-dilute to 10 ml with 1 mM Na2HCO3
-centrifuge 4500g for 10 minutes (5500 rpm JA17)
-the top ½ to 1/3 of pellet resuspended in 2-3 ml of supernatant
-mix with 10 ml of 81% Sucrose. Layer in bottom of tube
-layer with 3.5 ml of 53.4%, 48%, 46%, 44%, 42.6%, 40% Sucrose respectively
-centrifuge in SW28 at 25000 rpm for 90 minutes
-the plasma membrane is collected form 40-42.6% interface (top interface). The pellet of gradient contains the nuclei.
-dilute plasma membrane with 10 mM Tris, pH 7.5
-spin 20 minutes at 7750 rpm in JA17
-resuspend in 10 mM Tris, pH 7.5 and spin again
-resuspend in 800 ul of 10 mM Tris, pH 7.5. Aliquot in 200 ul fractions and spin in microfuge at 14 000 rpm for 25 minutes. Remove supernatant and freeze pellet in –80. Remember to take 25 ul for protein assay.
Nuclei Isolation
-the pellet from the plasma membrane sucrose gradient contains the nuclei
-resuspend the pellet in 20 ml of 10 mM Tris, pH 7.5
-centrifuge at 7750 rpm for 20 minutes in JA17 rotor. Repeat
-resuspend pellet in 2 ml 10 mM Tris, pH 7.5. Aliquot into 200 ul fractions and spin in microfuge at 14000 rpm for 25 minutes. Remove supernatant and freeze pellets at –80. Remember to take 25 ul for protein assay.
Mitochondria Isolation
-mitochondria are found in the pellet from the first ER spin
-resuspend pellet in 20 ml of 10 mM Tris, pH 7.5
-centrifuge at 7750 rpm for 20 minutes in JA17. Repeat
-resuspend pellet in 2 ml 10 mM Tris, pH 7.5. Aliquot into 200 ul fractions and spin in microfuge at 14000 rpm for 25 minutes. Remove supernatant and freeze pellets at –80. Remember to take 25 ul for protein assay.