Description

This Nuclear/Cytosol Extraction Kit provides a complete system that enables the separation of nuclear extract from the cytoplasmic fraction of mammalian cells. The optimized reagents and procedures provided with the kit allow separation of nuclear and cytoplasmic fractions quickly with little or no cross-contaminations. The extracted nuclear and cytoplasmic protein fractions are functional and compatible with downstream assays such as transcriptional activity, RNA splicing, gel shift assay, reporter assays, enzyme activity assays, and Western blotting.

Datasheet

FAQ

Can we use frozen cells?
No,freezing will break up the nucleus.

What kind of protein yield you can get using the kit?
Around 50-100 ug nuclear protein extract.

Where are the membrane, ER, mitochondrial, etc. proteins?
All are in cytosol.

What should I do if my sample is too dilute?
Spin-filter at 3500 RPM. This will get rid of water and the salts it contains and also the glycerol which is part of nuclear fraction.

What should I do if the cells clumped when I vortexed them in step 8?
This happens often. That is why step 9 says "Repeat step 8 every 10 minutes". What also helps is if you break the clump by pipetting up and down several times.

What Can I do with contaminations between the two fractions?
There are 3 things the customer can do to reduce contamination: 1. Make sure all the cells are suspended well in the CEB-A mix before adding CEB-B by combination of pipette and vortex, so that all cells are lysed by CEB-B. Some cells tend to associate together, over-trypsinization also can make the cells associate together. 2. Completely remove the cytosol fraction by a quick spin to remove any leftover cytosol fractions in the nuclei. 3. If contamination is still a issue, wash the nuclei with PBS once or twice before extracting the nuclei.

Can we obtain the native chromatin through this kit or only the total protein?
You can isolate only the protein. At step 7, you obtained the whole nuclei (including chromatin and all nuclear proteins). However, at the final step, you obtained the total soluble nuclear protein extracts. The chromatin bound insoluble proteins have been precipitated at step 10. How does the nuclear/cytosol seperation occur?
The CEB-A helps in suspending the cells. CEB-B is detergent which can lyse the cell membrane, but not the nuclear. NEB extracts the nuclear fraction.

Why do I have some ppt. in my reaction?
This precipitate is due to a small amount of protease inhibitor precipitating when diluted in buffer. Either spin down to remove this precipitate, or do not remove the precipitate and continue the experiment as is. It will cause no adverse effect.

Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.

Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.

What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.

Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.

What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.

Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.

How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.

Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.

Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.


CITATION
1. Hanzhong Ke. Interaction of PIAS1 with PRRS virus nucleocapsid protein mediates NF-κB activation and triggers proinflammatory mediators during viral infection. Sci Rep, July 2019; 31363150.
2. Zixiang Zhu. Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation. J Virol, July 2019; 31167907.
3. Xinran Li. Deregulated Gab2 phosphorylation mediates aberrant AKT and STAT3 signaling upon PIK3R1 loss in ovarian cancer. Nat Commun., Feb 2019; 30755611.
4. Huang-Ju Tu. The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells. Clin Epigenetics, Dec 2018; 30594243.
5. Lakkyong Hwang, Scolopendra subspinipes mutilans Extract Suppresses Inflammatory and Neuropathic Pain In Vitro and In Vivo. Evid Based Complement Alternat Med, Dec 2018; 30647762.

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