Cytoplasmic and Nuclear Protein Extraction

November 30, 2015

Texas Tech University Health Science Center
Internal Medicine
Postdoctoral Researcher

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Company:

Thermo Scientific

Product Name:

NE-PER Nuclear and Cytoplasmic Extraction Reagents

Catalog Number:

78833

This Kit is used to prepare the cytoplasmic and nuclear extracts from cultured cells and also in tissue. We can prepare active non-denatured proteins in less than two hours. It is mainly used in the EMSA and also in variety of applications like western blot, reporter assay, BCA protein assays and enzyme activity assays.

Experimental Design and Results Summary

Application

EMSA by using native polyacrylamide gel

Starting Material

Mouse cell lysate

Protocol Overview

Harvest the cells and centrifuge about 500xg for 5 min. Wash the cells and suspend it in PBS. Centrifuge again at 500xg for 2 min and remove the supernatant and add ice cold CER I to the pellet. Vortex vigorously on the highest setting and incubate on ice for 10 min. Add ice cold CER II, vortex again for 5 seconds, and centrifuge briefly for 16000xg for 5 min. Transfer the supernatant (cytoplasmic extract) into a new tube. Suspend the insoluble fraction in ice cold NER. Vortex it every 10min and incubate on ice for a total of 40 min. Centrifufge the tube at 16000xg for 10 min and collect the supernatant in a new tube.

Tips

Maintain the ratio of CER I:CER II:NER reagents 200:11:100ul

Results Summary

This kit is efficient to isolate the active proteins particularly for EMSA. The first two reagents CER I and CER II cause cell membrane disruption and release the cytoplasmic contents. After recovering the intact nuclei from centrifugation, the proteins were extracted out of the nuclei using NER.

Additional Notes

None

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Summary

The Good

This product generally has less than 10% contamination between nuclear extracts and cytoplasmic fractions.

The Bad

None

The Bottom Line

This kit is very helpful in isolating nuclear extracts for a variety of applications. Generally whole cell lysates affect the nuclear protein interactions and stabilty. Nuclear proteins were more concentrated in the nuclear extracts than in whole cell lysates. This product is highly recommended for EMSA and super shift EMSA assays.

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