Materials and Reagents

Sucrose
Protease Inhibitor cocktail, EDTA free (Merck KGaA, Calbiochem, catalog number: 539134 )
Phosphatase inhibitor cocktail II
Phosphatase inhibitor cocktail III
PMSF at 10 mM in DMSO or 1.74 mg/ml (Thermo Fisher Scientific, catalog number: 36978 )
Phosphatase Arrest II cocktail (Geno Technology, catalog number: 786-451 )
Phosphatase Arrest III cocktail (Geno Technology, catalog number: 786-452 )
M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, catalog number: 78501 )

Equipment

Sterile bottle filter
Glass Dounce homogenizer
Refrigerated centrifuge
Microfuge tubes rated for at least 25,000 x g centrifugation

Procedure

Make a 0.25 M sucrose solution that contains protease inhibitor cocktail, phosphatase inhibitor cocktails II and III, and PMSF as follows:
Per 100 μl of Sucrose master mix add:
a. 96 μl of 0.25 M sucrose
b. 1 μl of protease inhibitor cocktail
c. 1 μl of phosphatase inhibitor cocktail II
d. 1 μl of phosphatase inhibitor cocktail III
e. 1 μl of PMSF
Weigh tissue to be analyzed and add 10x its mass in volume of sucrose master mix (see step 1; i.e. 100 mg = 1,000 μl of sucrose solution).
While keeping all solutions on ice, add the appropriate amount of sucrose solution to tissue and dounce homogenize until a completely homogenous solution is obtained.
Spin the homogenate at 10,000 x g for 10 min at 4 °C.
Transfer the supernatants to a new microfuge tube (save the pellet at -20 °C) and spin at 30,000 x g for 90 min in a fixed angle rotor (or at 25,800 x g for 2 h).
Transfer the supernatant to a different microfuge tube and save at -20 °C. The remaining pellet corresponds to the microsomal fraction.
Pipette gently to resuspend the microsome pellet in 200 μl of the following mix (per 100 μl):
a.96 μl of MPER buffer
b.1 μl of protease inhibitor cocktail
c.1 μl of phosphatase arrest cocktail II
d.1 μl of phosphatase arrest cocktail III
e.1 μl of PMSF

Acknowledgments

We thank Dr. Gene Ness, Dr. Huntington Potter, and Dr. Chad Dickey for supporting the development and adaptation of this protocol in their labs. We credit the following article for this work: Abisambra et al. (2013). Financial support during the time of protocol development came from the Alzheimer’s Association NIRGD-12-242642, the Foundation for PSP/CBD and Related Brain Disorders (6144107400), and NIH/NIA ADC Pilot Grant from 5P30AG028383-08.

References

Abisambra, J. F., Jinwal, U. K., Blair, L. J., O'Leary, J. C., 3rd, Li, Q., Brady, S., Wang, L., Guidi, C. E., Zhang, B., Nordhues, B. A., Cockman, M., Suntharalingham, A., Li, P., Jin, Y., Atkins, C. A. and Dickey, C. A. (2013). Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J Neurosci 33(22): 9498-9507.
Abisambra, J. F., Fiorelli, T., Padmanabhan, J., Neame, P., Wefes, I. and Potter, H. (2010). LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease. PLoS One 5(1): e8556.