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9038
Cell Fractionation Kit
Experimental Controls
Buffer Kit

Cell Fractionation Kit #9038

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To Purchase # 9038S
Product # Size Price
9038S
1 Kit  (20 assays) N/A

Product Includes Quantity
Cytoplasmic Isolation Buffer (CIB) 10 ml
Membrane Isolation Buffer (MIB) 10 ml
Cytoskeletal/Nuclear Isolation Buffer (CyNIB) 5 ml
Protease Inhibitor Cocktail (100X) 5871 250 µl

Storage

Store at -20°C. Upon receipt, Protease Inhibitor Cocktail 5871 should be stored at 4°C. All other components should be stored at -20°C.

Protocol

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Cell Fractionation Kit Protocol

A. Buffers

  • Cytoplasm Isolation Buffer (CIB) – 10 ml, Store at -20°C.
  • Membrane Isolation Buffer (MIB) – 10 ml, Store at -20°C.
  • Cytoskeleton/Nucleus Isolation Buffer (CyNIB) – 5 ml, Store at -20°C.
  • Protease Inhibitor Cocktail (100X) (#5871) – 250 µl, Store at 4°C.

B. Notes

  • All steps, except for the addition and sonication of the CyNIB buffer, should be done on ice or at 4°C.
  • Adherent or suspension cultured cells can be used for this assay.
  • 1X protease inhibitors (#5871) (5 μl of 100X per 500 μl buffer, included in kit) and 1 mM fresh PMSF (#8553) (2.5 μl of 200 mM PMSF per 500 μl buffer, not included in kit) should be added to each buffer immediately before use.
  • Phosphatase inhibitors are already included in the buffers. There is no need to add them.
  • Please refer to Table 1 for the appropriate volumes for your specific cell concentration.
  • The volumes given in Sections D and E are based on cell counts obtained from HeLa cells at ~90% confluency in a 10 cm cell culture dish (5 x 106 cells).
  • If the CyNIB buffer is cloudy after thawing, please warm the solution in a 37°C water bath until the solution is clear.
  • Be cautious when saving fractions so that you do not get any contamination from the resulting pellet.
  • All lysates should be stored at -20°C for short term storage (less than 1 month) or -80°C for long term storage (greater than 1 month).

C. Isolating Cell Population

For adherent cells

  1. Wash plate with cold 1X PBS.
  2. Trypsinize the plate.
  3. Add cold growth media to deactivate trypsin.

For both adherent and suspension cells

  1. Spin down cells at 350 x g for 5 min.
  2. Aspirate media.
  3. Wash cell pellet with cold 1X PBS.
  4. Resuspend pellet in 0.5 ml of cold 1X PBS.
  5. Count live cells using Trypan Blue and a hemacytometer.

D. Detection of Proteins

  1. Aliquot 100 µl of cell suspension into a 1.5 ml tube for the whole cell lysate (WCL).
  2. Add 60 μl of 3X SDS Loading Buffer with DTT (#7722) to make a final volume of 160 µl of WCL.
  3. Sonicate WCL tube for 15 sec at 20% power 3 times, heat for 5 min at 95°C, and centrifuge for 3 min at 15,000 x g.

E. Cell Fractionation

  1. Aliquot the remaining 400 µl into a 1.5 ml tube.
  2. Centrifuge for 5 min at 500 x g at 4°C.
  3. Aspirate the supernatant.
  4. Resuspend pellet in 500 μl of CIB.
  5. Vortex for 5 sec.
  6. Incubate on ice for 5 min.
  7. Centrifuge for 5 min at 500 x g.
  8. Save the supernatant. This is the Cytoplasmic Fraction.
  9. Resuspend pellet in 500 μl of MIB.
  10. Vortex for 15 sec.
  11. Incubate on ice for 5 min.
  12. Centrifuge for 5 min at 8,000 x g.
  13. Save the supernatant. This is the Membrane and Organelle Fraction.
  14. Resuspend pellet in 250 μl of CyNIB.
  15. Sonicate for 5 sec at 20% power 3 times. This is the Cytoskeletal and Nuclear Fraction.

F. Western Blot

  1. Add 60 μl of 3X SDS Loading Buffer with DTT (#7722) for every 100 μl of supernatant.
  2. Boil each sample for 5 min at 95°C and centrifuge for 3 min at 15,000 x g.
  3. Load 15 µl of each fraction along with 15 µl of WCL.

Table 1: Volumes in μl for WCL or buffer at indicated cell numbers.

Cell Count
2.5 x 106 cells 5 x 106 cells 7.5 x 106 cells 1 x 107 cells
WCL 50 100 150 200
CIB 250 500 750 1000
MIB 250 500 750 1000
CyNIB 125 250 375 500

Protocol Id: 584

Product Description

The Cell Fractionation Kit is designed to provide a fast and efficient way of separating cultured cells into three distinct fractions: cytoplasmic, membrane/organelle, and nuclear/cytoskeletal. These fractions can then be analyzed by SDS-PAGE and western blotting. The kit includes enough buffer for 20 assays.

Background

Cellular fractionation allows for the extraction of cellular proteins into distinct compartments. This is achieved by the use of detergents that take advantage of the inherent qualities and composition of different cellular membranes (1). Cellular fractionation has been important for defining the localization of many proteins, observing the translocation of proteins, and determining protein-protein complexes, such as cytoskeletal-associated proteins (2,3). Thus, detergent-based cellular fractionation separates cellular components with greater ease and speed compared to a more laborious density centrifugation method (4).

  1. Lenstra, J.A. and Bloemendal, H. (1983) Eur J Biochem 135, 413-23.
  2. Banno, A. et al. (2012) J Biol Chem 287, 13799-812.
  3. Loo, L.H. et al. (2009) J Cell Biol 187, 375-84.
  4. Michelsen, U. and von Hagen, J. (2009) Methods Enzymol 463, 305-28.

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