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ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit
Isolation of high quality cytoplasmic and nuclear proteins 

#K0311	for 50 preps
Related Documents (in pdf, ~200 KB): Detailed Protocol: #K0311 MSDS (English) MSDS (English-USA) MSDS (German) Flyer

 

Features

• Versatile - isolates intact nuclei and cytoplasmic protein fraction from the same cell or tissue sample.
• High quality fractionation - no cross-contamination between cytoplasmic and nuclear fractions.
• Fast - procedure takes less than one hour.
• Compatible with downstream applications:
  • protein quantification,
  • 1D and 2D electrophoresis,
  • immunoprecipitation and Western blotting,
  • EMSA, enzymatic activity and reporter gene assays.

Description
The ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit is designed for rapid stepwise isolation of native, non-denatured cytoplasmic proteins and intact nuclei from cultured cells or tissue samples. The procedure, which is based on lysis with mild detergents, is fast and simple.
The isolated proteins are ready for direct use in many proteomic or enzymatic activity applications, including immunoprecipitation, affinity purification and reporter gene assays. Cell extracts prepared using this kit are compatible with common downstream applications, including 1D and 2D electrophoresis, Western blotting and electrophoretic mobility shift assays (EMSA). Isolated proteins are also compatible with quantification assays such as Bradford, Lowry and the BCA.
The extracted nuclei can either be stored frozen in the provided Nuclei Storage Buffer or lysed with Nuclei Lysis Reagent. The intact nuclei further may be used for isolation of chromatin, histones and nuclear RNA. Nuclear extracts are used in DNA-protein interaction assays (EMSA), 2D fractionation or other applications.
The kit contains sufficient reagents for extraction of cytoplasmic and nuclear fractions from 50 samples of 50 µl wet cell pellet or 100 mg of tissue.

Application
Preparation of native, non-denatured nuclear and cytoplasmic protein extracts from mammalian cultured cells or tissue samples.

Quality Control
The kit is functionally tested for cytoplasmic and nuclear protein extraction from cultured mammalian suspension cells.

Components of the Kit

• Cell Lysis Buffer
• Nuclei Washing Buffer
• Nuclei Storage Buffer
• Nuclei Lysis Reagent
• 1 M DTT
• Detailed Protocol

Storage
All components of the kit, except 1 M DTT, can be stored at 4°C for at least one year. Store 1 M DTT in aliquots at -20°C.

Figure 1. Cytoplasmic and nuclear proteins extracted using the ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit from various cell lines.
10 µg of cytoplasmic proteins and 5 µl of purified nuclei were separated in 12% SDS-PAGE, gel stained with PageBlue™ Protein Staining Solution.
1 - cytoplasmic proteins from Jurkat cells
2 - nuclear proteins from Jurkat cells
3 - cytoplasmic proteins from HeLa cells
4 - nuclear proteins from HeLa cells

Figure 2. EMSA of cytoplasmic and nuclear proteins extracted from various cell lines.
* [gama³³-P] labeled DNA containing AP-1 transcription factor binding sequence.
1 - no protein added
2 - cytoplasmic proteins from Cos7 cells
3 - nuclear proteins from Cos7 cells
4 - cytoplasmic proteins from NIH3T3 cells
5 - nuclear proteins from NIH3T3 cells
6 - cytoplasmic proteins from HeLa cells
7 - nuclear proteins from HeLa cells

Figure 3. Western blot analysis demonstrating pure cytoplasmic and nuclear protein fractions.
5 µg of cytoplasmic protein and 10 µl of purified nuclei were separated in 12% SDS-PAGE. The cytoplasmic protein tubulin and nuclear protein histone H4 were detected using specific antibodies.
1 - cytoplasmic proteins from Jurkat cells
2 - nuclear proteins from Jurkat cells
3 - cytoplasmic proteins from HeLa cells
4 - nuclear proteins from HeLa cells

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