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#OP-0002-1 EpiQuik Nuclear Extraction Kit
Product name : EpiQuik Nuclear Extraction Kit
Catalog number : OP-0002-1
Quantity: 100 assays
Supplier name : EpigenTek
Data sheet: Ask more or other datasheet now !
More Details about
OP-0022-100 EpiQuik Nuclear Extraction Kit II (Nucleic Acid-Free) (100 extractions)
The EpiQuik™ Nuclear Extraction Kit provides the simple and selective method for extracting nuclear proteins to be used for a variety of applications. These applications may include western blotting, protein-DNA binding assays, nuclear enzyme assays, and any other procedures requiring optimization or nucleic acidfree nuclear proteins. The EpiQuik™ Nuclear Extraction Kits are also specifically designed to meet the requirements of nuclear extracts used in various EpiQuik™ assays. The EpiQuik™ Nuclear Extraction Kits can be used to extract nuclear proteins from mammalian cells and tissue samples. The EpiQuik™ Nuclear Extraction Kits include kit I for regular nuclear extraction and kit II for nucleic acid-free nuclear extraction. The EpiQuik™ Nuclear Extraction Kit has the fastest procedure available on the current market, which can be completed within 60 minutes.
PRODUCT USE INFORMATION
The EpiQuik™ Nuclear Extraction Kit is very suitable for quick preparation of nuclear extracts from mammalian cells and tissue samples.
The EpiQuik™ Nuclear Extraction Kit is for research use only and is not intended for diagnostic or therapeutic application.
Epigentek guarantees the performance of all products in the manner described in
our product instructions.
Epigentek reserves the right to change or modify any product to enhance its
performance and design.
EpiQuik™ is a trademark of Epigentek Group Inc.
Components 100 extractions OP-0002-1
NE1 (10X pre-extraction buffer) 10 ml
NE2 (extraction buffer) 10 ml
1000X DTT solution 110 μl
1000X protease inhibitor cocktail (PIC) 110 μl
SHIPPING AND STORAGE
Store all components at 4°C. The kit is stable for up to 1 year from date of shipment when stored properly.
Cell Pellet Preparation
For Monolayer or Adherent Cells:
1. Grow cells to 70-80% confluence on a culture plate or flask (about 2-5 X 106 cells for a 100 mm plate). Remove the growth medium and wash cells with PBS twice and then remove PBS.
2. Add 1 ml of fresh PBS per 20 cm2 area (e.g., add 3 ml of PBS to a 100 mm plate), and scrape cells into a 15 ml conical tube. (Alternative option: detach cells with trypsin/EDTA and collect cells into a 15 ml conical tube. Count cells in a hemacytometer.)
3. Centrifuge the cells for 5 min at 1000 rpm and discard the supernatant.
4. Dilute NE1 with distilled water at a 1:10 ratio. Add DTT solution and PIC to ice cold diluted NE1 (1X) at a 1:1000 ratio. Re-suspend cell pellet in 100 μl of diluted NE1 per 106 cells and transfer to a micro centrifuge vial.
5. Incubate on ice for 10 min. Vortex vigorously for 10 sec and centrifuge the preparation for 1 min at 12,000 rpm.
6. Carefully remove the cytoplasmic extract from the nuclear pellet.
For Suspension Cells:
1. Grow cells to 2 X 106/ml and collect the cells into a 15 ml conical tube.
2. Centrifuge the cells for 5 min at 1000 rpm and discard the supernatant. Wash cells with PBS once by centrifugation for 5 min at 1000 rpm. Discard the supernatant.
3. Dilute NE1 with distilled water at a 1:10 ratio. Add DTT solution and PIC to ice cold diluted NE1 (1X) at a 1:1000 ratio. Re-suspend cell pellet in 100 μl of diluted NE1 per 106 cells and transfer to a micro centrifuge vial.
4. Incubate on ice for 10 min. Vortex vigorously for 10 sec and centrifuge
the preparation for 1 min at 12,000 rpm.
5. Carefully remove the cytoplasm extract from the nuclear pellet.
For Tissue Samples:
1. Weigh tissue and cut it into small pieces. Place cut pieces in a clean homogenizer. 2. Dilute NE1 with distilled water at a 1:10 ratio. Add 5 ml of diluted NE1 (1X) containing 5 μl of DTT per gram of tissue and homogenize tissue pieces (50-60 strokes).
3. Incubate on ice for 15 min and centrifuge for 10 min at 12,000 rpm at 4°C. Remove the supernatant.
Nuclear Extract Preparation
1. Add DTT solution and PIC to NE2 at a 1:1000 ratio. Add 2 volumes of NE2 containing DTT and PIC to nuclear pellet (about 10 μl per 106 cells or per 2 mg tissues). Incubate the extract on ice for 15 min with vortex (5 sec) every 3 min. The extract (especially tissue extract) can be further sonicated for 3 X 10 sec to increase nuclear protein extraction.
2. Centrifuge the suspension for 10 min at 14,000 rpm at 4°C and transfer the supernatant into a new microcentrifuge vial.
3. Measure the protein concentration of the nuclear extract.
4. Use immediately or aliquot and freeze the supernatant at –80°C until further use. Avoid freeze/thaw cycle.
1. Should DTT be added into NE1 for tissue extraction?
It is not necessary.
2. How many rpm should be used for centrifuging the cell extract if using desk-top centrifuge?
3. What should I do if addition of 5 µl NE2 into 1 M of cells can not suspend the nuclei?
Add more NE2, Ex: 10 µl to 1 M nuclei.
4. Can more solution be added for assay reaction if more nuclear extracts will be added?
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WP1099: Nuclear receptors in lipid metabolism and toxicity
WP1184: Nuclear Receptors
WP1326: Nuclear receptors in lipid metabolism and toxicity
WP1385: Nuclear Receptors
WP139: Nuclear receptors in lipid metabolism and toxicity
WP1502: Mitochondrial biogenesis
WP170: Nuclear Receptors
WP1845: MAPK targets/ Nuclear events mediated by MAP kinases
WP217: Nuclear Receptors
WP299: Nuclear receptors in lipid metabolism and toxicity
WP431: Nuclear receptors in lipid metabolism and toxicity
WP509: Nuclear Receptors
WP755: Nuclear receptors in lipid metabolism and toxicity
WP831: Nuclear Receptors
WP863: Nuclear receptors in lipid metabolism and toxicity
WP950: Nuclear Receptors
WP981: Nuclear receptors in lipid metabolism and toxicity
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