Description

This FractionPREP™ Cell Fractionation system is designed to provide reproducible extraction of four subcellular protein fractions (Cytosol, nucleus, membrane/particulate, and cytoskeletal fractions) from a single mammalian sample. The method is fast and simple, needing only 2 hours and no ultracentrifugation involved. All four protein fractions obtained are suitable for many downstream applications such as 1-D or 2-D gel, enzyme activity assays, gel shift assay, and Western blotting.

Datasheet

FAQ

If I don't need the particulate, can I exclude the intermediate steps?
Unfortunately, there is no way to skip the membrane extraction step. The buffers have the necessary detergents to remove the nuclear membrane

What components does each of the isolated fractions contains?
Nuclear Fraction: Total nucleus soluble proteins, including the nuclear membrane proteins.Cytosol Fraction: Total cellular soluble proteins from cytoplasm. Membrane/Particulate: Total cellular membrane proteins including cellular organelles and organelle's membrane proteins (but excluding the nuclear membrane proteins).Cytoskeletal Fraction: Total cellular insoluble proteins, genomic DNA.

In western analysis, a membrane protein appeared mainly in his nuclear fraction. Why?
The membrane extraction step may not be sufficient. First, make sure to add Membrane Extraction Buffer B into the extraction, which contain NP-40 detergent. If it still shows the same result, increase incubation time from 1 min to 5 min after adding MEB-B. NP-40 should extract membrane from most of cells.

Do you have antibodies for use in conjunction with the each fraction?
Here are some of the antibodies that can be used with all of our Cell Fractionation Kits:
Cytosol Fraction: 3189-100 Anti-Calpain; 3138-100 Anti-Caspase-3 pAb
Nuclear Fraction: 3342-100 Anti-Oct-1 pAb
Mitochondrial Fraction: 3026-100 Anti-Cytochrome c mAb; 3025-100 Anti-Cytochrome c pab
Membrane Fraction: 3365-100 Anti-Cadherine pAb

Could you let us know the percentage of four protein fractions by using this product?
Percentage of proteins in the different fractions are cell-dependent, usually in the range of ~20-30% cytosol, ~25-35% membrane (organelles), ~15-25% Nuclei, ~15-25% insoluble.

Does the last obtained fraction contain DNA? Should DNase be added in the sample?
DNA will be in the last fraction, and small amount DNA can be in nuclear extract fraction. If you really concern about the presence of DNA, you can certainly add DNase. However, for most applications, it might be fine without DNase treatment.

Can frozen samples be used for fractionation?
We have not used frozen samples here at BioVision. However, customer has reported that frozen samples work fine in customer’s protein localization studies.

How much are the concentration of the salts in the fractions?
The nuclear fraction has high salt concentration. The other ones have medium level similar to the physiological buffers.

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.

Which fraction will have the intracellular vesicles, endosomes, lysosomes, etc.?
This kit is based on the sequential fractionation and isolation of subcellular proteins by using various detergents in the Extraction buffer to yield different fractions. Since endosome and lysosomes are subcellular organelles that are membrane bound, they will be in the membrane fraction.

In the FAQ section of the kit it says that DNA is in the last fraction, together with the cytoskeletal proteins. Is it possible to isolate the DNA from the proteins in this fraction?
We do not have a protocol for isolating the DNA from the proteins in the above mentioned fraction. However, generally if you want to remove DNA from protein solution, you can do a Polyethyleneimine (PEI) precipitation. You can add PEI to your protein solution to a final concentration of 0.02%. Stir on ice for a half hour while the nucleic acids are precipitated from solution. Centrifuge to remove the precipitated material and the supernatant can be used for any downstream applications. Other options will be to treat your samples with DNases.

CITATIONS

1. Chao, Min-Wu et al. (2017) Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells, Sci Rep. 2017 Apr 7;7:46134.
2. Ying et al., GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway. J. Mol. Endocrinol., Oct 2015; 55: 245 - 262.
3. Chen et al., Acyl-CoA-binding domain containing 3 modulates NAD+ metabolism through activating poly(ADP-ribose) polymerase 1. Biochem. J., Jul 2015; 469: 189 - 198.
4. Cheng et al., Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate. J. Biol. Chem., Jul 2014; 289: 20871 - 20878.
5. Pérez et al., Metabolic Rescue of Obese Adipose-Derived Stem Cells by Lin28/Let7 Pathway. Diabetes, Jul 2013; 62: 2368 - 2379.

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