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In molecular biology and cell biology experiments, high-purity extraction of nuclear proteins is a key step in studying nuclear processes such as gene regulation, transcription factor activity, and chromatin structure. Due to the difference in distribution between nuclear proteins and cytoplasmic proteins, in order to ensure experimental data accuracy, a nuclear protein extraction method with low cross-contamination and easy operation is required to obtain high-purity nuclear protein samples. In addition, nuclear proteins are sensitive to the external environment, and special attention should be paid to operating conditions during the extraction process, such as temperature control and the use of protease inhibitors, to maintain protein activity and avoid degradation.

It is particularly important to choose the appropriate nuclear protein extraction method for different experimental needs. For example, for proteins highly expressed in the nucleus such as HIF1-α and TWIST, it is recommended to use a nuclear protein extraction kit or a dedicated nuclear protein lysis buffer for extraction. These methods not only optimize the operation steps, but also effectively reduce the cross-contamination between cytoplasm and nuclear proteins, providing a reliable sample basis for subsequent experiments (such as Western Blot, immunoprecipitation, etc.).

Here are two relatively easy-to-use nuclear protein extraction methods:

For proteins with high expression in the nucleus, such as HIF1-α and TWIST, a special nuclear protein extraction kit or nuclear protein lysis buffer should be used for extraction.

Nuclear protein extraction steps (homogenization grinding method)

Preparation

Before the nuclear protein extraction experiment, relevant reagents and buffers need to be prepared in advance to ensure that the experimental process proceeds smoothly. The following is the detailed formula and usage of Trypan Blue dye solution and buffer:

Trypan Blue dye solution formula

Preparation of mother solution: Weigh an appropriate amount of Trypan Blue powder and dissolve it in 10 mL of double distilled water to prepare a 4% (w/v) Trypan blue mother solution.

Working solution dilution: Use PBS buffer to dilute the mother solution 10 times to obtain a 0.4% concentration working solution.

Usage: Mix 0.4% Trypan blue working solution and cell suspension at a volume of 1:1 for cell counting or cell activity detection.

Buffer A formula (cell lysis buffer)

Ingredients:

10 mM HEPES (pH 7.9, 4°C)

1.5 mM MgCl₂

10 mM KCl

0.5 mM DTT (dithiothreitol)

If you need to prevent the degradation of plasma proteins from affecting nuclear proteins, you can add 1 mM PMSF (phenylmethylsulfonyl fluoride)

Function:

Used for cell membrane lysis, release of cell nuclei, and ensure the integrity of the cell nucleus.

Buffer B formula (nuclear protein extraction buffer)

Ingredients:

20 mM HEPES (pH 7.9)

25% (v/v) glycerol

0.42 M NaCl

1.5 mM MgCl₂

0.2 mM EDTA (ethylenediaminetetraacetic acid)

0.5 mM PMSF

0.5 mM DTT

Function:

Rupture the nuclear membrane and release nuclear proteins while maintaining the stability and activity of the proteins.

Nuclear protein extraction steps

Cell collection

● Digest the cells from the flask and collect them into micro tubes.

● Centrifuge at 300 g for 5 min at 4°C and dispose the supernatant.

● Wash the Cell precipitation 3 times with PBS buffer to ensure that residual impurities are removed.

Preparation for cell lysis

● After centrifugation, dispose the supernatant. Add 5 times the volume of pre-cooled Buffer A (for example, add 100 μL Buffer A for every 20 μL cell precipitation), pipette to mix, and fully suspend the cells.

● Place the cell suspension on ice for 10 min, then centrifuge at 300 g for 5 min at 4°C.

Nucleus release

● After centrifugation, discard the supernatant, add 25 times the volume of pre-cooled Buffer A again, and gently pipette to resuspend the cells.

● Transfer the cell suspension to a Dounce homogenizer for homogenization.

Homogenization monitoring

● During the homogenization process, take a small amount of cell suspension and mix it with an equal volume of 0.4% Trypan blue dye.

●Observe the cell membrane rupture under a microscope:

●If most cells are stained blue, it means that the cell membrane has been ruptured, and stop homogenization.

●If it is not completely ruptured, continue homogenization.

Separation of cell nucleus and cytoplasm

●When most cells are ruptured, transfer the cell suspension to an Eppendorf tube and centrifuge at 25,000 g for 20 min at 4°C.

Centrifugation results:

●The supernatant contains cytoplasmic components.

●The precipitate is the cell nucleus, which contains nuclear protein.

Nuclear protein extraction

●Collect the precipitate after centrifugation, add 1 volume of pre-cooled Buffer B, and gently blow to resuspend the precipitate.

●Transfer the suspension to a clean tissue homogenizer and homogenize 30 times to ensure that the nuclear protein is fully released.

Protein dissolution and extraction

●Transfer the homogenized suspension to an Eppendorf tube and incubate it in a low-speed shaker on ice for 45 min to ensure that the nuclear protein is fully dissolved.

●Centrifuge at 25,000 g for 30 min at 4°C and collect the supernatant, which is the purified nuclear protein.

Nuclear protein extraction steps (kit method)

Preparation

Reagent preparation

●Equilibrate the nuclear protein extraction kit to room temperature.

●Add PMSF to the plasma protein extraction reagent and nuclear protein extraction reagent to a final concentration of 1 mM.

●If the sample contains cysteine, add DTT to both reagents to a final concentration of 0.5 mM.

Cell collection and washing

●Digest the cells from the flask wall and collect them in an Eppendorf tube.

●Centrifuge at 300 g for 5 min at 4°C and discard the supernatant.

●Wash the cells 1-2 times with PBS buffer to remove residual impurities.

Cell lysis

● Centrifuge at 300 g for 10 min at 4°C and discard the supernatant.

● Add 5 volumes of plasma protein extraction reagent (e.g., add 100 μL per 20 μL cell pellet) and gently mix by inverting to ensure that the cells are evenly suspended. Be careful to avoid vortexing to prevent excessive cell disruption.

Lysis reaction

● Place the cell suspension on ice for 20 min to completely lyse the cells.

Cytoplasmic protein separation

● Centrifuge at 250 g for 20 min at 4°C and discard the supernatant.

● Add 2 volumes of pre-cooled plasma protein extraction reagent to the cell pellet and mix gently.

Nuclear release

● Use a syringe with a 0.14 mm needle to gently blow 5 times to fully lyse the cell pellet.

● Centrifuge at 10,000 g for 30 min at 4°C, collect the supernatant (cytoplasmic protein) and discard the pellet (nuclear protein).

Nuclear protein extraction

●Add 50-100 μL of nuclear protein extraction reagent to the nuclear protein precipitate, and gently blow 5 times with a 0.14 mm diameter needle syringe to ensure that the precipitate is fully suspended.

●Place the suspension on a low-speed shaker on ice and incubate for 45 minutes to promote the dissolution of nuclear protein.

Nucleoprotein separation

●Centrifuge at 16,000 g for 5 minutes at 4°C and collect the supernatant, which is the extracted cell nuclear protein.

Tissue nuclear protein extraction steps

Tissue sample preparation

●Weigh the tissue sample to be extracted.

●Cut the tissue into small pieces as possible to increase the homogenization efficiency.

Tissue homogenization

●Add 300-500 μL PBS for every 50 mg of tissue.

●Use a homogenizer to fully homogenize under ice bath conditions to ensure that the tissue is completely dissociated and forms a uniform suspension.

Centrifugation and precipitate collection

●Centrifuge at 500 g for 3 min at 4°C and discard the supernatant in the tissue suspension.

●Collect the precipitate, which contains cells and organelles.

Cell nuclear extraction

●Add the collected precipitate to 200 μL plasma protein extraction solution.

●Continue to extract cell nuclear protein according to the above centrifugation steps.

The selection of a suitable method should be determined based on the experimental objectives, sample size, and experimental equipment. In practical applications, researchers can flexibly select a suitable nuclear protein extraction method based on their own needs and the specific characteristics of the sample. No matter which method to choose, ensuring the accuracy of the operation and low temperature conditions is the key to obtaining high-quality nuclear proteins.

I hope this article can provide valuable references for everyone, help optimize the nuclear protein extraction process, and improve the reliability and repeatability of the experiment.