It is possible that the nuclear and cytoplasmic extracts can be used for a Western blot. The kit is designed to maintain the functional activity of proteins for assays such as EMSAs (with non-denaturing PAGE) where the protein structure should remain relatively intact. For Western blots, proteins are linearized using denaturing PAGE gels, typically with the presence of SDS detergent or other denaturants. To proceed, it is suggested to use RIPA buffer as the extraction buffer/dilution buffer for each fraction separately after separating the cytoplasmic fraction from the nuclear fraction.
NXTRACT
NuCLEAR™ Extraction Kit
For mammalian tissue or cultured cells
Synonym(s):
Nuclear Isolation Kit
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About This Item
UNSPSC Code:
41105517
NACRES:
NA.56
Pricing and availability is not currently available.
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Quality Level
usage
kit sufficient for 10 extractions (1 ml packed cell volume)
kit sufficient for 100 extractions (100 μl packed cell volume)
technique(s)
protein extraction: suitable
western blot: suitable
shipped in
dry ice
storage temp.
−20°C
General description
The procedure for the nuclear protein extraction method is to allow cells to swell with hypotonic buffer. The cells are then disrupted, the cytoplasmic fraction is removed, and the nuclear proteins are released from the nuclei by a high salt buffer.
Application
Other Notes
Within this kit is a complete system for preparing nuclear and cytoplasmic protein extracts from mammalian tissue or cultured cells. All reagents necessary for extraction are included.
A number of different procedures in the detailed technical bulletin enable the selection that best fits a particular application. For example, choose between detergent and non-detergent extraction of nuclear protein or between the standard hypotonic lysis buffer for most cell types and isotonic lysis buffer for fragile cells. In addition, the kit provides a procedure for salt reduction from the nuclear extract with dilution buffer. NuCLEAR offers the flexiblity you need for optimal protein extraction. Extracts can be prepared in less than 2 hours and are highly pure since there is little or no cross-contamination between nuclear and cytoplasmic extracts.
Legal Information
NuCLEAR is a trademark of Sigma-Aldrich Co. LLC
Kit Components Also Available Separately
Product No.
Description
SDS
- 3× Dilution and Equilibration Buffer 90 mL
- P8340Protease Inhibitor Cocktail 1 mLSDS
Signal Word
Danger
Hazard Statements
Precautionary Statements
Hazard Classifications
Eye Dam. 1 - Met. Corr. 1 - Skin Corr. 1A
Storage Class Code
8A - Combustible corrosive hazardous materials
Flash Point(F)
188.6 °F - closed cup
Flash Point(C)
87 °C - closed cup
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Articles
Centrifugation separates organelles based on size, shape, and density, facilitating subcellular fractionation across various samples.
Centrifugation separates organelles based on size, shape, and density, facilitating subcellular fractionation across various samples.
Centrifugation separates organelles based on size, shape, and density, facilitating subcellular fractionation across various samples.
Centrifugation separates organelles based on size, shape, and density, facilitating subcellular fractionation across various samples.
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Is it possible to use the cytoplasmic and nuclear fractions for Western Blotting?
1 answer-
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Can Product NXTRACT, CelLytic™ NuCLEAR™ Extraction Kit, be used to make nuclear protein extracts for EMSAs/DNA binding studies?
1 answer-
Several references describe the use of the N-XTRACT kit in sample preparation for EMSA work.1. Xie, J., et al., β2-Microglobulin as a negative regulator of the immune system: high concentrations of the protein inhibit in vitro generation of functional dendritic cells. Blood, 101(10), 4005-4012 (2003).2. Marambaud, P., et al., A CBP Binding Transcriptional Repressor Produced by the PS1/ε-Cleavage of N-Cadherin Is Inhibitedby PS1 FAD Mutations. Cell, 114, 635-645 (2003).3. Chen, G., et al., Phosphorylated FADD induces NF-κB, perturbs cell cycle, and is associated with poor outcome in lung adenocarcinomas. Proc. Nat. Acad. Sci. USA, 102(35), 12507-12512 (2005).
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Is Product NXTRACT, CelLytic™ NuCLEAR™ Extraction Kit, suitable for use on tissues?
1 answer-
We have used the kit with several tissues: Mouse lung and brain, Rat liver and Rabbit muscle. For these tissues we usually used the kit with 100 mg tissue. After extraction with 140 μl Extraction buffer, we obtained an average yield of protein concentration as follows: 3.2 mg/ml for brain, 4.1 mg/ml for lung, 3.3 mg/ml for muscle and 10 mg/ml for liver.
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What is the Department of Transportation shipping information for this product?
1 answer-
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
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How "pure" is the nuclear extraction using Product NXTRACT, CelLytic™ NuCLEAR™ Extraction Kit?
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It is important to keep in mind that this kit will provide a crude nuclear fraction. Depending on the care taken at the step where the nuclei are pelleted and the supernatant containing the cytoplasmic fraction is removed, we have found that the cytoplasmic contamination of the nuclear pellet may be a few percent.
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When using Product NXTRACT, CelLytic™ NuCLEAR™ Extraction Kit, what can I use for the mechanical lysis of my cells?
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We recommend the use of a glass tissue homogenizer, with a type B pestle, or alternatively a syringe with a narrow-gauge (No. 27) hypodermic needle may be used.
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What are the options for lysis of my cells to make extracts when using Product NXTRACT, CelLytic™ NuCLEAR™ Extraction Kit?
1 answer-
The use of detergent will lead to maximum protein yield. However we find that the hypotonic lysis is extremely effective and in addition for very fragile cells we offer an isotonic lysis option.
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