CelLytic™ NuCLEAR™ Extraction Kit. SIGMA/NXTRACT – For mammalian tissue or cultured cells. Product Type: Chemical. Application A number of different procedures in the detailed technical bulletin enable the selection that best fits a particular application. For example, choose. CelLytic NuCLEAR Extraction Kit Product Code NXTRACT TECHNICAL BULLETIN Product Description The preparation of an extract from nuclei is often the first.
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The resulting preparation can be used directly in the Electrophoresis Mobility Shift Assay EMSAfootprinting analysis, transcription assays, or as a starting point for the purification of regulatory proteins. 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. Nuclear proteins can be extracted from the following fresh or frozen tissues: The protein extracts are suitable for the detection of DNAprotein interaction using a gel-shift assay, DNase I footprinting analysis, and similar techniques.
Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. The DTT solution and the protease inhibitor cocktail must be stored at 20 C and should be added to buffers immediately before use.
All the buffer solutions may be stored at 2 8 C for several weeks. Procedure Perform all steps at 2 8 C. Use precooled buffers and equipment. Ensure all the solutions are defrosted and homogeneous. All centrifugations are done at 4 C with precooled rotors. The final concentration of DTT in the solutions should be 1 mm. The protease inhibitor cocktail should be diluted fold in the final solutions. Dilute the 1 M DTT solution with deionized, sterile water to a concentration of 0.
Prepare 1X Lysis Buffer, hypotonic, from the 10X Lysis Buffer, hypotonic, by diluting fold with sterile, deionized water. Remove the growth medium from the cells. Rinse the cells twice with PBS, being careful not to dislodge any of the cells.
Scrape the cells using fresh PBS into an appropriate conical centrifuge tube. Cells in suspension Collect the cells into an appropriate conical centrifuge tube. Wash cells twice by resuspending the cell pellets in PBS and centrifuge for 5 minutes at x g. Estimate the packed cell volume PCV. Resuspend the cell pellet gently. If working with small volumes, the suspended cells may be transferred to a microcentrifuge tube.
Incubate the packed cells in the selected lysis buffer on ice for 15 minutes, allowing cells to swell. Take several microliters of the cells in the lysis buffer and view them under the microscope.
If massive cell lysis is detected under the microscope or a gelatinous mass is observed, the cells may be fragile. In this case, use the Lysis Buffer, isotonic, for cell lysis and consider eliminating the incubation step. Vortex vigorously for 10 seconds. Centrifuge immediately for 30 seconds at 10, x g.
In order to assess the degree of lysis, before centrifugation, take a sample of the suspended cells and view the nuclei under the microscope. Lysis can be observed by the addition of the Trypan Blue solution to an aliquot of cells. The dye is excluded from the intact cells, but stains the nuclei of lysed cells. If lysis of nuclei is observed under the microscope or if a gelatinous mass is observed, lyse the cells with a lower final concentration of IGEPAL CA Avoid vortexing the cells and centrifuge at a slower speed.
Transfer the supernatant to a fresh tube.
TECHNICAL BULLETIN. CelLytic NuCLEAR Extraction Kit. Product Code NXTRACT – PDF
If it is necessary to extract the proteins of interest at a lower salt concentration, dilute the Extraction Buffer with 1X Dilution and Equilibration Buffer. The salt concentration in nuclearrtm Extraction Buffer is 0.
In rare cases a lower or a higher salt concentration may be needed for a better nuclaertm of a particular protein. Mount the tube on a vortex mixer and agitate at medium to high speed for minutes. Centrifuge for 5 minutes at 20, x g. Transfer the supernatant to a clean, chilled tube. Snap-freeze the supernatant in aliquots with liquid B. Detergents can interfere with the activity or binding of the extracted proteins. Therefore, a procedure for nuclear protein extraction without the use nculeartm a detergent is included.
This protocol describes the preparation of crude nuclear extracts using a syringe or a glass tissue homogenizer. Use of a syringe is recommended for small-scale preparations 0. Passage of more than one millliliter through a syringe may cause difficulties due to the needle gauge size. Dilute the 1 M DTT solution to 0. Prepare 1X Lysis Buffer, hypotonic. For protein extraction from fragile cells, prepare 1X Lysis Buffer, isotonic, to replace the 1X Lysis Buffer, hypotonic.
Cells in suspension Collect the cells into an appropriate centrifuge conical tube. Decant and discard supernatant. Incubate the packed cells in lysis buffer for 15 minutes, allowing cells to swell.
Centrifuge the suspended cells for 5 minutes at x g. Using a glass tissue homogenizer, transfer the cells into a glass tissue grind tube.
Grind on ice slowly with five up-and-down strokes using a type B pestle. Using a syringe with a narrow-gauge No. The syringe plunger is used to displace the buffer as fully as possible. This removes all the air from the syringe and prevents excess air being pumped into the cell suspension during lysis.
TECHNICAL BULLETIN. CelLytic NuCLEAR Extraction Kit. Product Code NXTRACT
Draw the cell suspension nucleagtm into the syringe and then eject with a single rapid stroke. The number of strokes needed using the tissue homogenizer or the syringe varies between cell lines. Start with 5 strokes and then check lysis under the microscope.
If the lysis is not sufficient, perform several more strokes until lysis is complete. If nuclear lysis or clumps of nuclei are visualized, or if a gelatinous mass is observed, the cell disruption was too vigorous or too many strokes were performed.
Centrifuge the disrupted cells in suspension for 20 minutes at 10, 11, x g. If the proteins of interest are extracted at a lower salt concentration, dilute the Extraction Buffer with the 1X Dilution and Equilibration Buffer. If the procedure is being performed with a tissue homogenizer, it is nuclezrtm to give 10 more strokes at this point. Shake gently for 30 minutes. Snap-freeze the supernatant in aliquots with liquid C.
Nuclear protein extraction from mg of tissue Calculate accordingly for extraciton tissue weight. Prepare 1X Lysis Buffer For tissues tested by Sigma the hypotonic buffer works better than the isotonic. Therefore, it is recommended to prepare 1X Lysis Buffer, hypotonic.
If the tissue is found to be too fragile, one can use the 1X Lysis Buffer, cellhtictm. Rinse the tissue twice with PBS buffer. If one wishes to extract nuclei proteins with a lower salt concentration, the Extraction Buffer may be diluted with 1X Dilution and Equilibration buffer. At this stage a short homogenization can be performed to facilitate nuclear extraction. Centrifuge for 5 minutes at 20, 21, x g. Snap-Freeze the supernatant in aliquots with liquid D.
Salt Removal The nuclear proteins extracted according to the protocol are suspended in Extraction Buffer, a high salt buffer. Usually the proteins extracted are highly concentrated and can be diluted with a low salt buffer 1X Dilution extarction Equilibration Buffer.
Since small amounts of the concentrated extract in a high salt buffer are sufficient for analysis by EMSA, footprinting, and similar assays, the salt dilution occurs naturally in the reaction tube itself. If salts interfere with nuleartm experiments, removal of salts may be performed rapidly using desalting gel-filtration columns see Reagents and Equipment Recommended for Salt Removal. The protease inhibitor cocktail should be added to the eluted protein fraction.
The salts may also be removed by dialysis of the nuclear extracts extrqction a dialysis buffer that is similar to the 1X Dilution and Equilibration Buffer, containing a final concentration of 1 mm DTT and protease inhibitor cocktail or 0. Sephadex is a registered trademark of Amersham Biosciences Ltd.
Purchaser must determine the suitability of the product s cellytjctm their particular use. Additional terms and conditions may apply. extrqction
Please see reverse side of the invoice or packing slip. Benchtop Mitochondria Isolation Protocol Note: Specific protocols are available fxtraction the following products: An antibody for the protein of interest is incubated with a cell extract so that the antibody.
Thawing Human Cell Lines for culture: Locate vials in liquid nitrogen and quickly remove with forceps. Take vial to sterile. PR G-Biosciences technical gbiosciences. MCR Connect with Epicentre on our blog epicentral. Please read these instructions carefully The viability of these cells is warranted for 30 days from date of shipment when specified reagents and growth conditions are followed as described in. Box Rockford, IL