Welcome guest, Sign in

Home

X
加载中

This protocol details the extraction of microsomes from frozen tissue in order to further examine the protein-protein interactions occurring within the endoplasmic reticulum. This protocol was adapted from Abisambra et al. (2013) with modifications made in order to optimize for subsequent use.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Microsome Isolation from Tissue

Cell Biology > Organelle isolation > Microsome
Authors: Maria Bodero
Maria BoderoAffiliation: Sanders-Brown Center on Aging and Department of Physiology, University of Kentucky, Lexington, USA
Bio-protocol author page: a1139
 and Jose Francisco Abisambra
Jose Francisco AbisambraAffiliation: Sanders-Brown Center on Aging and Department of Physiology, University of Kentucky, Lexington, USA
For correspondence: joe.abisambra@uky.edu
Bio-protocol author page: a1140
Vol 4, Iss 3, 2/5/2014, 2552 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1038

[Abstract] This protocol details the extraction of microsomes from frozen tissue in order to further examine the protein-protein interactions occurring within the endoplasmic reticulum. This protocol was adapted from Abisambra et al. (2013) with modifications made in order to optimize for subsequent use.

Keywords: Endoplasmic reticulum, Microsomes, Sub-cellular fractionation

Materials and Reagents

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

Equipment

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

Procedure

  1. 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
  2. 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).
  3. 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.
  4. Spin the homogenate at 10,000 x g for 10 min at 4 °C.
  5. 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).
  6. Transfer the supernatant to a different microfuge tube and save at -20 °C. The remaining pellet corresponds to the microsomal fraction.
  7. 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

  1. 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.
  2. 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.


How to cite this protocol: Bodero, M. and Abisambra, J. F. (2014). Microsome Isolation from Tissue. Bio-protocol 4(3): e1038. DOI: 10.21769/BioProtoc.1038; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook