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Cell and Nuclear Extraction Protocols |
Introduction
Cell and nuclear extraction are vital techniques to isolate cellular components for analysis. These methods are applicable to both adherent and suspension cells. Adherent cells are scraped off their growth surface, while suspension cells are collected by centrifugation. Both types of cells undergo lysis and centrifugation to yield cytoplasmic and nuclear fractions. These high-quality extracts are essential for various research applications, including protein quantification and signal transduction studies.
This protocol has been developed for rapid isolation of cytokines and signaling molecules, using a non-abrasive extraction reagent for total protein extraction. The method is sensitive and allows for the detection of disease-associated biomarker fluctuations. The prepared extracts are compatible with a variety of immunoassays, including ELISA and the Luminex platform, as well as conventional protein assays. Typically, this extraction method is cost-effective, versatile, and can be completed in less than 15 minutes.
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Materials
- Microcentrifuge tubes (Pierce Microcentrifuge Tubes, 1.5 mL)
- Conical tubes(Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes)
- Cell extraction buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate)
Note: 1X Cell extraction buffer may be divided into 1X aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting cells.
Additional recommended materials
- Protease inhibitors (Halt Protease Inhibitor Cocktail (100X) and PMSF Protease Inhibitor)
- DMSO, Anhydrous
- Phosphate-buffered saline (20X PBS)
- Protein Assays (Quant-iT Protein Assay Kit)
Protocol tips
Addition of the protease inhibitor cocktail and PMSF is necessary to inhibit proteolysis in cell extracts. Just prior to use, supplement the cell extraction buffer with 1 mM PMSF and protease inhibitor to make complete cell extraction buffer. Prepare 0.3 M PMSF stock in DMSO and add final concentration of 1 mM PMSF (i.e., 17 μL of 0.3M PMSF stock in 5 mL cell extraction buffer). PMSF is very unstable and must be added just before use, even if it was added previously. For Protease Inhibitor addition, we recommend using Halt Protease Inhibitor Cocktail, reconstituted to 1X (10 μL of 100X Halt Protease Inhibitor Cocktail in 1 mL of cell extraction buffer). The stability of protease inhibitor-supplemented cell extraction buffer is 24 hours at 4°C.
Procedure
Processing cell culture samples
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Processing cell lysates
Note: This method can be used to produce relatively large quantities of cell extracts with each of the stimulation regimes studied. The wash steps included in this procedure help to minimize medium components in the cell extracts.
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Materials
- Microcentrifuge tubes (Pierce Microcentrifuge Tubes, 1.5 mL)
- Hypotonic buffer solution (20 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2)
- NP-40 Surfact-Amps Detergent Solution
- Phosphate-buffered saline (PBS, pH 7.2)
- Cell extraction buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate)
Note:1X Cell extraction buffer may be divided into 1X aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting cells.
Additional recommended materials
- Protease inhibitors (Halt Protease Inhibitor Cocktail (100X) and PMSF Protease Inhibitor)
- DMSO, Anhydrous
- Protein Assays (Quant-iT Protein Assay Kit)
Protocol tips
To inhibit proteolysis in cell extracts, it is essential to add a protease inhibitor cocktail and PMSF. Just before use, supplement the cell extraction buffer with 1 mM PMSF and Protease Inhibitor to prepare complete cell extraction buffer. Prepare 0.3 M PMSF stock in DMSO and add it to achieve a final concentration of 1 mM PMSF (i.e., 17 μL of 0.3 M PMSF stock in 5 mL cell extraction buffer). PMSF is very unstable and must be added immediately before use, regardless of prior addition. For the Protease Inhibitor, we recommend using Halt Protease Inhibitor Cocktail, reconstituted to 1X (10 μL of 100X Halt Protease Inhibitor Cocktail in 1 mL of cell extraction buffer). The stability of cell extraction buffer supplemented with protease inhibitor is 24 hours at 4°C.
Procedure
Note: This protocol has been successfully applied to several cell lines of human origin. Researchers should optimize the nuclear extraction procedure for their own applications.
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Microplate readers
Stylesheet for Classic Wide Template adjustments
For Research Use Only. Not for use in diagnostic procedures.