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ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit

Nuclear and cytoplasmic extract from Hela cells were collected using ReadiPrep™ Nucleus/Cytosol Fractionation Kit (AAT Bioquest®, Cat#60000). The protein was quantified by Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit (AAT Bioquest®, Cat#. 11100). <strong>A</strong>. 8 µg of nuclear or cytoplasmic extract was incubated with or without HDAC inhibitor Trichostatin A, and HDAC activity was measured by Amplite® Fluorimetric HDAC Activity Assay Kit (AAT Bioquest®, Cat#. 13601). <strong>B</strong>. 40 ug total protein of nuclear or cytoplasmic extract was used.  2 ug/ml of rabbit anti-HDAC1 antibody was used to probe the nitrocellulose membrane for overnight. 10 ug/ml of iFluorTM 647 goat anti-rabbit IgG (H+L) (AAT Bioquest®, Cat#. 16809) was used. The detection was visualized by UVP MultiSpectral Imaging System (Biolite). M: Marker, Nu: Nuclear extract, Cyto: Cytoplasmic extract.
Nuclear and cytoplasmic extract from Hela cells were collected using ReadiPrep™ Nucleus/Cytosol Fractionation Kit (AAT Bioquest®, Cat#60000). The protein was quantified by Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit (AAT Bioquest®, Cat#. 11100). <strong>A</strong>. 8 µg of nuclear or cytoplasmic extract was incubated with or without HDAC inhibitor Trichostatin A, and HDAC activity was measured by Amplite® Fluorimetric HDAC Activity Assay Kit (AAT Bioquest®, Cat#. 13601). <strong>B</strong>. 40 ug total protein of nuclear or cytoplasmic extract was used.  2 ug/ml of rabbit anti-HDAC1 antibody was used to probe the nitrocellulose membrane for overnight. 10 ug/ml of iFluorTM 647 goat anti-rabbit IgG (H+L) (AAT Bioquest®, Cat#. 16809) was used. The detection was visualized by UVP MultiSpectral Imaging System (Biolite). M: Marker, Nu: Nuclear extract, Cyto: Cytoplasmic extract.
Nuclear and cytoplasmic extract from Jurkat cells were collected using ReadiPrep™ Nucleus/Cytosol Fractionation Kit. The protein was quantified by Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit (AAT Bioquest<sup>®</sup>, Cat#. 11100). 8 µg of nuclear or cytoplasmic extract was incubated with or without HDAC inhibitor Trichostain A, and HDAC activity was measured by Amplite® Fluorimetric HDAC Activity Assay Kit (AAT Bioquest®, Cat#. 13601).
Nuclear and cytoplasmic extract from Hela cells were collected using ReadiPrep™ Nucleus/Cytosol Fractionation Kit. The protein was quantified by Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit (AAT Bioquest<sup>®</sup>, Cat#. 11100). 8 µg of nuclear or cytoplasmic extract was incubated with or without HDAC inhibitor Trichostain A, and HDAC activity was measured by Amplite® Fluorimetric HDAC Activity Assay Kit (AAT Bioquest®, Cat#. 13601).
Nuclear and cytoplasmic extract from CHO cells were collected using ReadiPrep™ Nucleus/Cytosol Fractionation Kit. The protein was quantified by Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit (AAT Bioquest<sup>®</sup>, Cat#. 11100). 8 µg of nuclear or cytoplasmic extract was incubated with or without HDAC inhibitor Trichostain A, and HDAC activity was measured by Amplite® Fluorimetric HDAC Activity Assay Kit (AAT Bioquest®, Cat#. 13601).
40 ug total protein of nuclear or cytoplasmic extract from Jurkat cells was used.  2 ug/ml of mouse anti-α-tubulin antibody or rabbit anti-HDAC1 antibody was used to probe the nitrocellulose membrane for overnight. 10 ug/ml of iFluor<sup>TM</sup> 647 goat anti-mouse IgG (H+L) (AAT Bioquest®, Cat#. 16744) or iFluor<sup>TM</sup> 647 goat anti-rabbit IgG (H+L) (AAT Bioquest®, Cat#. 16809) was used. The detection was visualized by UVP MultiSpectral Imaging System (Biolite). M: Marker, Nu: Nuclear extract, Cyto: Cytoplasmic extract.
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Catalog Number60000
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


The ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit offers stepwise isolation of nuclear and cytoplasmic extract from mammalian cells or tissue. The isolated proteins have high concentrations and preserve their biological activities. The nuclear and cytoplasmic extracts from ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit are compatible with many downstream applications including enzyme activity assays and fluorescent Western blotting etc.

Components


Component A: Cytosol Extraction Buffer1 Bottle (25 mL)
Component B: Nuclear Extraction Buffer1 Bottle (8 mL)
Component C: 10X Protease Inhibitors1 Vial (4 mL)
Component D: DTT 1 Vial (100 uL)

Example protocol


AT A GLANCE

Protocol summary

  1. Rinse cells with PBS
  2. Add 500 µL of Cytosol Extraction Buffer
  3. Centrifuge for 20 seconds
  4. Collect the supernatant (Cytoplasmic extract)
  5. Re-suspend the pellets in 150 µL 1X High Salt Buffer
  6. Centrifuge for 20 minutes
  7. Collect the supernatant (Nuclear Extract)

Important notes
Thaw all the kit components at room temperature before starting the experiment (you may store buffers at 4°C or –20°C), keep the buffers on ice during the experiment.

PREPARATION OF WORKING SOLUTION

1. Cytosol Extraction Buffer (1X):
Add 100 µL of 10X Protease Inhibitors (Component C) and 1 µL of DTT (Component D) into 0.9 mL of Cytosol Extraction Buffer (Component A).

2. Nuclear Extraction Buffer (1X):
Add 100 µL of 10X Protease Inhibitors (Component C) and 1 µL of DTT (Component D) into 0.9 mL of Nuclear Extraction Buffer (Component B).

Note: 0.5 mL 1X Cytosol Extraction Buffer and 150 µL of 1X Nuclear Extraction Buffer is enough for 1 assay, prepare fresh buffer as needed.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Rinse the cells 1 time with cold PBS. For cells in suspension, collect the cells by centrifugation.

  2. Add 500 µL of 1X Cytosol Extraction Buffer to cells. For adherent cells, scrape the adherent cells into a 1.5 mL centrifuge tube.

  3. Vortex vigorously to fully re-suspend the cells.

  4. Centrifuge at 16,000 g for 1 - 2 minutes and transfer the supernatant (Cytoplasmic extract) to another clean tube. Keep the tube on ice for downstream applications or store at -80°C.

  5. Re-suspend the pellet in 150 µL 1X Nuclear Extraction Buffer.

  6. Vortex vigorously to fully re-suspend the pellet.

  7. Rotate the tube at 4°C for 30 minutes.

  8. Centrifuge at 16,000g for 20 minutes and transfer the supernatant (Nuclear extract) to another clean tube. Keep the tube on ice for downstream applications or store at -80°C.

Citations


View all 2 citations: Citation Explorer
Circ\_001422 aggravates osteosarcoma progression through targeting miR-497-5p/E2F3 axis
Authors: Li, Xinyu and Zhao, Xin and Li, Jin and Zhang, Xiaozhan
Journal: (2022)
Hsa\_circ\_0088212-mediated miR-520 h/APOA1 axis inhibits osteosarcoma progression
Authors: Liu, Feng and Zhang, Xiangyang and Wu, Fei and Peng, Hao
Journal: Translational Oncology (2021): 101219

References


View all 2 references: Citation Explorer
Studied by Rapid Fractionation of Barley (Hordeum vulgare) Protoplasts
Authors: Gardestrom, P.; Wigge, B., Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, undefined
Journal: Plant Physiol (1988): 69-76
and mitochondria
Authors: Lilley, R. M.; Stitt, M.; Mader, G.; Heldt, H. W., Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol
Journal: Plant Physiol (1982): 965-70