SOP for Nuclear and Cytoplasmic Fractionation

Utility: This protocol allows western-blotting based measurements of the distribution of your protein of interest between the nucleus and cytoplasm of cells. This is useful for studying proteins involved in nuclear translocation or shuttling by other proteins.

Notes: Actin can be used as a housekeeping gene as it is present in both fractions, but if you want cytoplasmic and nuclear-specific housekeeping genes Histone H3 and GAPDH are good options. Make sure to check if these are equally expressed between cell lines which may be compared, as alterations in expression between lines could confound results.

Intro: This is a protocol I've developed using the Thermo Fisher NE-PER Kit, #78833. The protocol has been modified from that recommended by the manufacturer, as my lab identified some issues with cross-contamination between nuclear and cytoplasmic fractions. These were evidenced by GAPDH presence in the nuclear pellet. Let me know if there's anything I should modify on this to make it more clear.

Cell Culture Preparation:

For adherent cells, harvest from T75 with 1 mL Trypsin-EDTA, quench with 4mL media, collect into 5 mL tube then centrifuge at 500 × g for 5 minutes
Dump out media and wash cells by re-suspending the cell pellet with 4 mL cold PBS
Spin down cells again 500 x g for 5 minutes
Dump out media and carefully remove supernatant, leaving pellet as dry as possible
Add ice-cold CER I to the cell pellet using volumes as follows and keep on ice:

Packed Cell Volume (uL)	CER I (uL)	CER II (uL)	NER (uL)
10	200	11	100
20	400	22	200
50	1000	55	500
100	2000	110	1000

Note: Pellet size can be approximated by preparing 5% milk in TBST and pipetting into a blank tube and comparing with cell pellet to visualize. Pipette in 10 uL to an empty version of the same tube type as your cell pellet and compare, then increase or decrease volume from there to approximate pellet size.

Cytoplasmic and Nuclear Protein Extraction

Note: Scale this protocol depending on the cell pellet volume. Maintain the volume ratio of CER I:CER II:NER reagents at 200:11:100µL, respectively.

Turn on microcentrifuge and fast temp to 4C
Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell pellet, and incubate on ice for 10 minutes
Add ice-cold CER II to the tube using volumes in above table
Vortex the tube for 5 seconds on the highest setting. Incubate tube on ice for 1 minute
Vortex the tube for 5 seconds on the highest setting.
Centrifuge the tube for 5 minutes at maximum speed in a microcentrifuge (~16,000 × g)
Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube, and retain tube containing pellet

Note: remove supernatant using gel loading tips to ensure pellet remains undisturbed. Pellet is translucent, move around using tip of pipette to visualize pellet

Mix together CER I and CER II in noted proportions below in a separate test tube and add to pellet as a wash. Vortex vigorously with pellet for 15 seconds

Note: Scale volume added in proportion to packed cell volume, using 50 uL CER I and 2.75 uL CER II for a 10 uL packed cell pellet and scaling accordingly

Remove CER I/CER II wash from pellet with gel loading tip and discard
Suspend the insoluble (pellet) fraction in ice-cold NER using volumes in above table
Break up pellet by sonicating 30 seconds, vortexing 15 seconds, and placing on ice for 5 minutes. Repeat process 8x or until pellet is completely solubilized
Centrifuge the tube at maximum speed (~16,000 × g) in a microcentrifuge for 10 minutes
Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice
Fractions are now separated and can be subjected to western blotting or stored at -20C for later analysis

Note: Prior to conducting SDS-PAGE analysis of fractions, thaw samples and run BCA assay to normalize protein loading in gel. This can be done by preparing BCA standards in equivalent buffers to nuclear and cytoplasmic fractions, followed by quantitation on a plate reader.

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SOP for Nuclear and Cytoplasmic Fractionation