Nuclear Extraction Protocol

In regards to life sciences and cell studies, nuclear protein extraction is the method of separating and isolating the nuclear portions of the cell (those from the nucleus) from the cytoplasmic portions of the cell. The process can be highly useful in sample preparation of the nuclear proteins especially in studies which look to study molecules that interact with the nucleus. Downstream applications for nuclear extraction include Western blot, protein-DNA binding assays, nuclear enzyme assays, and other procedures requiring optimized nuclear proteins.

The unique characteristic about nuclear extraction is that there are several methods in completing it to prepare the samples needed for the downstream procedures mentioned. From the default method which can be completed in just an hour, to extraction that needs to be nucleic acid-free, and even a related process for special applications like whole-cell extraction. EpiGentek provides the kits and tools you need to perform nuclear extraction, efficiently, quickly and cost-effectively.

See the various protocols involved with our different proprietary kits below, or click one of the links to jump to your preferred extraction method:

Standard Nuclear Protein Extraction Protocol

Kits involved: EpiQuik Nuclear Extraction Kit

The EpiQuik™ Nuclear Extraction Kit is a complete set of optimized reagents to provide a simple and selective method for isolating nuclear proteins used for a variety of applications in just 60 minutes. The kit is also specifically designed to meet the requirements of nuclear extracts used in various other epigenetic products from Epigentek, including DNMT, HDAC, and HAT assays. A total of 100 standard extractions (using 10⁷ cells or 20 mg tissues) can be performed with this kit. Total yield can be up to 100 µg per optimal extraction, although results may somewhat vary depending on the cell or tissue type.

EpiQuik Nuclear Extraction Kit Contents:

• PBS
• Trypsin/EDTA
• Desktop centrifuge (up to 14,000 rpm) capable of 4˚C
• Distilled or Deionized Water
• Vortex mixer
• Dounce homogenizer
• Sonication device
• 1.5 ml micro-centrifuge tubes

Protocol Cell Pellet Preparation

For Monolayer or Adherent Cells

1. Grow cells to 70-80% confluency in 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 minutes at 1000 rpm and discard the supernatant.
4. Dilute NE1 with distilled water at a 1:10 dilution (1X). Add DTT Solution and PIC to ice cold diluted NE1 (1X) at a 1:1000 dilution. Re-suspend cell pellet in 100 µl of diluted NE1 (1X) per 10⁶ cells and transfer to a micro-centrifuge vial.
5. Incubate on ice for 10 minutes. Vortex vigorously for 10 seconds then centrifuge the preparation for 1 minute at 12,000 rpm in a desktop centrifuge (about 11000 G).
6. Carefully remove the cytoplasmic extract from the nuclear pellet. (The cytoplasmic protein fraction may be quantified and used for downstream applications.)

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 minutes at 1000 rpm and discard the supernatant. Wash cells with PBS once by centrifugation for 5 minutes at 1000 rpm. Discard the supernatant.
3. Dilute NE1 with distilled water at a 1:10 dilution (1X). Add DTT Solution and PIC to ice cold diluted NE1 (1X) at a 1:1000 dilution. Re-suspend cell pellet in 100 µl of diluted NE1 (1X) per 10⁶ cells and transfer to a micro-centrifuge vial.
4. Incubate on ice for 10 minutes. Vortex vigorously for 10 seconds then centrifuge the preparation for 1 minute at 12,000 rpm.
5. Carefully remove the cytoplasmic extract from the nuclear pellet. (The cytoplasmic protein fraction may be quantified and used for downstream applications.)

For Tissue Samples

1. Weigh tissue and cut it into small pieces. Place the cut pieces in a clean homogenizer.
2. Dilute NE1 with distilled water at a 1:10 dilution (1X). Per gram of tissue, add 5 ml of diluted NE1 (1X) containing 5 µl of DTT and homogenize tissue pieces (50-60 strokes).
3. Incubate on ice for 15 minutes then centrifuge for 10 minutes 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 dilution. Add 2 volumes (based on pellet size) of NE2 containing DTT and PIC to nuclear pellet (about 10 µl per 10⁶ cells or per 2 mg of tissue). Incubate the extract on ice for 15 minutes with vortex (5 seconds) every 3 minutes. The extract (especially tissue extract) can be further sonicated for 3 x 10 seconds to increase nuclear protein extraction.
2. Centrifuge the suspension for 10 minutes at 14,000 rpm at 4°C and transfer the supernatant into a new micro-centrifuge vial.
3. Measure the protein concentration of the nuclear extract. *Note: A standard Bradford protein assay to measure the concentration of the nuclear extract is recommended. For accurate nuclear extract quantification, the final working buffer (NE2+PIC with or without DTT) should be used as a blank. After the protein concentration has been measured the DTT can be added for storage purposes.
4. Use immediately or aliquot and freeze the supernatant at -80°C until further use. Avoid multiple freeze/thaw cycles.

See more information, user guide & MSDS on the EpiQuik Nuclear Extraction Kit product page.

Nucleic-Acid Free Nuclear Protein Extraction Protocol

Kits involved: EpiQuik Nuclear Extraction Kit II (Nucleic Acid-Free)

The EpiQuik™ Nuclear Extraction Kit II (Nucleic Acid-Free) is a complete set of optimized reagents to provide a straightforward and selective process for isolating nuclear proteins free of nucleic acids attached, to be used in a range of downstream applications. Like the EpiQuik Nuclear Extraction Kit I, the entire process completes in just 60 minutes helping you save time to perform additional research.

EpiQuik Nuclear Extraction Kit II (Nucleic Acid-Free) Contents:

• PBS
• Trypsin/EDTA
• Desktop centrifuge (up to 14,000 rpm) capable of 4˚C
• Distilled or Deionized Water
• Vortex mixer
• Dounce homogenizer
• Sonication device
• 1.5 ml micro-centrifuge tubes

Protocol Cell Pellet Preparation

For Monolayer or Adherent Cells

1. Grow cells to 70-80% confluency 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 a100 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 minutes at 1000 rpm and discard the supernatant.
4. Dilute NP1 with distilled water at a 1:10 ratio (ex: 1 ml of NP1 + 9 ml of distilled water). Add DTT Solution and PIC to ice cold diluted NP1 (1X) at a 1:1000 ratio. Re-suspend cell pellet in 100 µl of diluted NP1 (1X) per 10⁶ cells, and transfer to a micro-centrifuge vial.
5. Incubate on ice for 10 minutes. Vortex vigorously for 10 seconds and centrifuge the preparation for 1 minute at 12,000 rpm.
6. Carefully remove the cytoplasmic extract from the nuclear pellet.

For Suspension Cells

1. Grow cells to 2 × 10⁶/ml and collect the cells into a 15 ml conical tube.
2. Centrifuge the cells for 5 minutes at 1000 rpm and discard the supernatant. Wash cells with PBS once by centrifugation for 5 minutes at 1000 rpm. Discard the supernatant.
3. Dilute NP1 with distilled water at a 1:10 ratio (ex: 1 ml of NP1 + 9 ml of distilled water). Add DTT Solution and PIC to ice cold diluted NP1 (1X) at a 1:1000 ratio. Re-suspend cell pellet in 100 µl of diluted NP1 (1X) per 10⁶ cells and transfer to a microcentrifuge vial.
4. Incubate on ice for 10 minutes. Vortex vigorously for 10 seconds and centrifuge the preparation for 1 minute at 12,000 rpm.
5. Carefully remove the cytoplasmic extract from the nuclear pellet.

For Tissue Samples

1. Weigh the tissue and cut it into small pieces. Place tissue pieces in a clean homogenizer.
2. Dilute NP1 with distilled water at a 1:10 ratio (ex: 1 ml of NP1 + 9 ml of distilled water). Add 5 ml of diluted NP1 (1X) containing 5 µl of DTT Solution per gram of tissue, and homogenize tissue pieces (50-60 strokes).
3. Incubate on ice for 15 minutes and centrifuge for 10 minutes at 12,000 rpm at 4°C. Remove the supernatant.

Nuclear Extract Preparation

1. Add DTT Solution and PIC to NP2 at a 1:1000 ratio, followed by adding NP3 to NP2 at a 1:10 ratio. Add 2 volumes of NP2 to nuclear pellet (about 10 µl NP2 per 10⁶ cells or per 2 mg of tissue). Incubate the extract on ice for 15 minutes with vortex (5 seconds) every 3 minutes. The extract (especially tissue extract) can be further sonicated for 3 x 10 seconds to increase nuclear protein extraction.
2. Centrifuge the suspension for 10 minutes at 14,000 rpm at 4°C and transfer the supernatant into a new microcentrifuge vial.
3. Add NP4 to the supernatant at a 1:100 ratio (ex: add 10 µl of NP4 to 990 µl of the supernatant and incubate for 15-20 minutes at room temperature.
4. Centrifuge the suspension for 1 minute at 14,000 rpm at 4°C and transfer the supernatant into a new microcentrifuge vial.
5. Measure the protein concentration of the nuclear extract
6. Use immediately or aliquot and freeze the supernatant at –80°C until further use. Avoid freeze/thaw cycle.

See more information, user guide & MSDS on the EpiQuik Nuclear Extraction Kit II (Nucleic Acid-Free) product page.

Whole Cell Protein Extraction Protocol

Kits involved: EpiQuik Whole Cell Extraction Kit

If instead of attempting to extract just the nuclear proteins from a cell and want to extract whole cell proteins, the EpiQuik™ Whole Cell Extraction Kit provides an easy and selective process for extracting whole cell proteins used for a range of applications. The EpiQuik™ Whole Cell Extraction Kit is also specifically designed to meet the requirements of whole cell extracts used in EpiQuik™ assays. Boasting a rapid 45-minute protocol, this kit has the fastest procedure available on the current market.

EpiQuik Whole Cell Extraction Kit Contents:

* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.

• PBS
• Trypsin/EDTA
• Desktop centrifuge (up to 14,000 rpm) capable of 4˚C
• Distilled or Deionized Water
• Vortex mixer
• 1.5 ml micro-centrifuge tubes

Protocol

Before starting, check if the 5X Extraction Buffer contains precipitates before using. If so, warm (at room temperature or 37°C) and shake or vortex the buffer until the precipitates are re-dissolved.

Cell Pellet Preparation

For Monolayer or Adherent Cells

1. Cells (treated or untreated) are grown to 80-90% confluency and trypsinized after removing growth medium. Cells are then collected into a 15 ml conical tube and counted in a hemacytometer.
2. Cells are washed once with PBS and pelleted by centrifugation at 1000 rpm for 5 minutes.

For Non-Adherent 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 minutes at 1000 rpm and discard the supernatant. Wash cells with PBS once by centrifugation at 1000 rpm for 5 minutes. Discard the supernatant.

Cell Extract Preparation

1. Prepare 1X Extraction Buffer by adding 1 ml of the 5X Extraction Buffer to 4 ml of distilled water.
2. Add 1000X DTT Solution and PIC to 1X Extraction Buffer at a 1:1000 ratio. Re-suspend cell pellet in 100 µl of ice cold 1X Extraction Buffer per 10⁶ adherent cells, or 2 x 10⁶ non-adherent cells.
3. Transfer the cell solution to a micro-centrifuge vial. Incubate on ice for 15 minutes with vigorous vortex (5 seconds) per 5 minutes.
4. Centrifuge the cell solution for 10 minutes at 14,000 rpm at 4ºC and transfer the supernatant into a new micro-centrifuge vial.
5. Measure the protein concentration of cell extract.
6. Use immediately or aliquot and freeze supernatant at –70ºC until further use. Avoid freeze/thaw cycle.

See more information, user guide & MSDS on the EpiQuik Whole Cell Extraction Kit product page.

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