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NE-PER Nuclear and Cytoplasmic Protein Extraction Reagents FAQ


Answers to frequently asked questions (FAQs) about NE-PER Nuclear and Cytoplasmic Protein Extraction Reagents


 

How do the NE-PER Reagents fractionate cellular proteins?
The CER I Reagent induces swelling of the cell causing stress on the cellular membrane. The CER II reagent gently lyses the cell membrane allowing cytoplasmic proteins to be collected, while leaving the nucleus intact. The NER Reagent is then used to extract nuclear proteins from the pellet.

Which downstream applications can I perform after protein extraction?
The following applications were successfully used with protein extracted using the NE-PER Reagents: electrophoretic mobility shift assay (EMSA), Western blotting, reporter assays, isoelectric focusing (IEF) and immunoprecipitation.

How can I determine the separation efficiency between the cytoplasmic and nuclear fractions?
Cross-contamination of the nuclear fraction by cytoplasmic proteins can be determined by probing for a protein known to reside only in the cytoplasm, such as a heat shock protein 90 (Hsp90), b-galactosidase or protein kinase C. Determine contamination of the cytoplasmic fraction by probing for Oct-1 or p53 nuclear proteins in the cytoplasmic fraction.

How can I minimize cross-contamination of proteins between the nuclear and cytoplasmic fractions? Typically, cross-contamination between the nuclear and cytoplasmic fraction is 10% or less when using this kit. Careful pipetting and performing the incubation and centrifugation as indicated in the instructions will minimize cross-contamination. Performing an additional wash of the nuclear pellet with phosphate buffer before adding the NER Reagent will reduce carryover of the cytoplasmic proteins to the nuclear protein fraction.

How much protein can I expect to recover from the nuclear and cytoplasmic fraction?
The total amount of protein recovered will vary with different sample types. Some examples are listed in the table below:

Cell/Tissue Type

Cytoplasmic Protein

Nuclear Protein

100mg of liver tissue

3 to 4mg

1 to 1.5mg

10^6 HeLa cells

300 to 400µg

40 to 60µg

10^6 monocytes

N/A

0.15 to 0.25µg

Which tissues and cell types have been used with the NE-PER Reagents?
We have extracted, soluble nuclear proteins from calf liver tissue, and cultured epithelial, fibroid, kidney, liver and brain cells. Many references have cited the use of various other cells and tissues with the NE-PER Reagents.

Can I use the NE-PER Reagents for both adherent and suspension cells?
Yes. The kit was optimized for a packed-cell volume; therefore, adherent cells require harvesting, either by scraping or trypsinization, from the culture flask for maximum recovery.

Can I use frozen cells or tissues?
Freezing and thawing will prematurely rupture cellular membranes and lead to high cross-contamination between the nuclear and cytoplasmic fractions.

Is the final soluble nuclear fraction dialyzable?
Yes. The nuclear fraction can be dialyzed in a Slide-A-Lyzer MINI Dialysis Unit (#69550) against a buffer, such as PBS, that is compatible with the specific downstream application. A buffer exchange can also be performed using the Pierce Protein Desalting Spin Columns (#89849) or Zeba Desalt Spin Columns (89882).

Have the NE-PER Reagents been tested with plant tissue or yeast cells?
No. Also, nuclear protein was not successfully isolated from yeast cells that were pre-lysed with Y-PER Yeast Protein Extraction Reagent.

Are there any considerations when adding protease inhibitors to the CER I and NER Reagents?
EDTA-free Halt Protease and/or Phosphatase Inhibitor Cocktails (e.g., #78425) can be added to CER I and NER reagents. Most commercially available protease inhibitor cocktails are also compatible; however, avoid protease inhibitors that contain alcohols. Because the concentration of the CER I and NER Reagents are critical, do not dilute these reagents by more than 5% when adding protease inhibitors.

After protein extraction, where is the genomic DNA located?
After extracting the nuclear proteins using the NER Reagent, genomic DNA will be located in the pellet.

How long does it take to complete the protocol?
Nuclear and cytoplasmic proteins can be isolated within 2 hours.

 

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Instructions | MSDS | CofA
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