The ab219177 Nuclear Extract Kit is a powerful tool for extracting nuclear proteins from mammalian cells or tissues in just 45 minutes. These nuclear proteins are essential for various applications, including western blotting and nuclear enzyme assays. In this blog post, we'll guide you through the protocol to make the process easy to understand and follow.
Nuclear protein isolation protocol
Materials You'll Need:
Before you start, gather the following materials:
The ab219177 Nuclear Extract Kit
1X Phosphate-buffered saline (PBS)
Trypsin/EDTA solution
Double-distilled water (ddH2O)
1.5 mL and 15 mL plastic tubes
Benchtop microcentrifuge
Centrifuge for 15 mL tubes
Sonicator
Step-by-Step Protocol:
Step 1: Preparation
◈ Ensure that PBS is at 4°C and store it on ice.
◈ Cool the benchtop microcentrifuge to 4°C.
◈ If you plan to use the extracts for enzyme activity assays, do not add Protease Inhibitor Cocktail to any buffers or fractions.
Step 2: Buffer Preparation
◈ For each extraction, transfer 500 µL each of Cytoplasmic Extraction Buffer, Nuclear Extraction Buffer, and Nuclear Lysis Buffer into clean 1.5 mL microcentrifuge tubes and keep them on ice.
◈ To each tube, add 2.5 µL of 200X Protease Inhibitor Cocktail and 2.5 µL of 200X DTT. Keep the tubes on ice until needed.
Cytoplasmic Extraction Buffer (++)
Nuclear Extraction Buffer (++)
Nuclear Lysis Buffer (++)
Step 3: Cell Preparation
◈ For adherent cells, grow cells to 70-80% confluence and remove the growth medium.
◈ Wash the cells with room temperature PBS twice.
◈ For suspension cells, grow cells to 2 x 106/mL.
◈ For tissues, weigh the tissue and cut it into small pieces for homogenization.
◈ Wash tissues twice with ice-cold PBS.
Step 4: Cell/Tissue Processing
◈ Follow specific instructions based on cell type (adherent, suspension, or tissues) for the next steps. These include resuspending the cells, centrifugation, and preparation for extraction. Please refer to the procedure for adherent cells inside the blue box below.
◈ Centrifuge for 5 minutes at 1,000 rpm (4°C) and discard the supernatant.
◈ Wash cells with 10 mL of ice-cold PBS by centrifugation for 5 minutes at 1,000 rpm (4°C) and discard the supernatant.
Step-by-Step Protocol for Adherent Cells:
a. Grow Adherent Cells
Cultivate your adherent cells on a culture plate or flask until they reach 70-80% confluency.
Remove the growth medium from the plate.
b. Wash the Cells
Wash the cells twice with room temperature PBS (Phosphate-buffered saline).
Carefully discard the PBS after each wash.
c. Collect the Cells
For every 20 cm2 of cell growth area, add 1 mL of room temperature PBS. (3 mL for 100 mm plate)
Use a cell scraper to gently detach the cells from the surface of the culture plate. Ensure all cells are in suspension.
d. Optional: Use Trypsin/EDTA
Alternatively, you can use trypsin/EDTA solution for detachment.
Dispense enough trypsin/EDTA solution to completely cover the monolayer of cells.
Incubate the cells in a 37°C incubator for approximately 2 minutes or until they detach from the surface.
Once detached, the cells will appear rounded.
e. Protect the Cells
Immediately after trypsinization, add serum or media containing serum to the cell suspension.
This helps protect the cells from any potential damage caused by the trypsin activity.
Note: It's important to be aware that the process of trypsinization may have an impact on the cellular pathway you are studying, so consider this when planning your experiments.
Step 5: Extraction of Cytoplasmic Proteins
◈ Resuspend the cell pellet in Cytoplasm Extraction Buffer (+/+) and transfer to a 1.5 mL tube.
◈ Vortex briefly and incubate cells on ice for 10 minutes.
◈ Vortex briefly again and centrifuge for 3 min at 1,000 g (4°C).
◈ Trasfer the supernatant (cytoplasmic protein extract) to new ice-cold 1.5 mL tube and keep both the pellet and the supernatant on ice.
Step 6: Extraction of Soluble Nuclear Proteins
◈ Resuspend the pellet from the previous step in Nuclear Extraction Buffer (++).
◈ Vortex briefly and incubate cells on ice for 15 minutes (with vortex every 5 min).
◈ Vortex briefly again and centrifuge for 3 min at 5,000 g (4°C).
◈ Trasfer the supernatant (soluble nuclear proteins "Nuclear Extract 1") to new ice-cold 1.5 mL tube and keep both the pellet and the supernatant on ice.
Step 7: Extraction of Insoluble Nuclear Proteins
◈ Resuspend the pellet from step 6 in Nuclear Lysis Buffer (++).
◈ Sonicate (low) the sample on ice to obtain "Nuclear Extract 2", which contains remaining insoluble nuclear proteins.
Step 8: Protein Quantification and Analysis
◈ Measure the protein concentration of the extracted fractions (BCA assay).
◈ Use the fractions immediately or aliquot and freeze at -80°C for future use.
Conclusion:
Using the ab219177 Nuclear Extract Kit, you can efficiently extract nuclear proteins from mammalian cells and tissues. This protocol simplifies the process into clear steps, making it accessible for your research needs.
