Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

This method can help answer key questions in the hypoxic field, such as endogenous and native interactions between transcription factors and their transcriptional coregulators under hypoxic conditions. The main advantage of this technique is that the hypoxic cells are harvested under hypoxic conditions and the nuclear fractions are used for the co-immunoprecipitation. This protocol uses human embryonic kidney 293A cells grown in four 10-centimeter dishes.

Seed three to five times 10 to the sixth cells per dish in 10 milliliters of Dulbeco's modified Eagle's medium supplemented with 10%FBS, L-glutamine, sodium pyruvate, penicillin and streptomycin. Culture the cells in a 37 degrees Celsius 5%carbon dioxide incubator. Twenty-four hours after seeding, when the cells have reached 80 to 90%confluency, put two dishes into the hypoxia subchamber in the incubator glovebox with 1%oxygen and 5%carbon dioxide at 37 degrees Celsius for four hours.

Keep the other two dishes at normoxia. On the day before harvesting the hypoxia-treated cells, pre-equilibrate DPBS to hypoxic conditions by placing an uncovered 100 milliliter experiment glass reagent bottle filled with DPBS in the hypoxia subchamber for 24 hours. Approximately one hour prior to harvesting the cells, place an icebox containing ice into the processing chamber of the glovebox, which has been equilibrated to 1%oxygen and 5%carbon dioxide.

Transfer the bottle containing the pre-equilibrated hypoxic DPBS from the hypoxia subchamber to the processing chamber and place it on ice. Four hours following hypoxia treatment, transfer the cells from the hypoxia subchamber to the processing chamber that has been pre-equilibrated to 1%oxygen and 5%carbon dioxide. Remove the culture medium by aspiration and with a 10 milliliter pipette, rinse the cells once with 10 milliliters of ice-cold pre-equilibrated hypoxic DPBS.

Using a five milliliter pipette, add 5 milliliters of ice-cold pre-equilibrated DPBS and dislodge the cells by scraping with a cell scraper. Tilt the cell culture plate, collect the detached cells using a 10 milliliter pipette and transfer the cell suspension into a 15 milliliter conical tube. Keep the tube of cells on ice.

Open the door between the processing and buffer chambers in the glovebox, both of which have been pre-equilibrated to 1%oxygen and 5%carbon dioxide. Transfer the 15 milliliter conical tubes on ice containing hypoxia-treated cells from the processing chamber to the buffer chamber. Open the door of the buffer chamber and remove the cells completely from the glovebox.

Pellet both the hypoxic cells and the normoxic cells by centrifugation at 1000 times g for five minutes at four degrees Celsius. A nuclear extraction kit will be used for this procedure. Start by gently resuspending each cell pellet in 500 microliters of lysis buffer NL, supplemented with 1X protease inhibitor cocktail in 0.1 molar DTT by pipetting up and down several times.

Add 25 microliters of detergent solution NP to the cell suspension and vortex for 10 seconds at maximum speed. Centrifuge at 1000 times 10 g at four degrees Celsius for five minutes. Save both the supernatant and the nuclear pellet.

After collecting and storing the supernatant as described in the text protocol, resuspend the pellet-containing cell nucleii in 500 microliters of lysis buffer NL, supplemented with 1X protease inhibitor cocktail and 0.1 molar DTT by vortexing for five seconds at maximum speed. Resuspend the nuclear pellet in 50 microliters of extraction buffer NX1, supplemented with 1X protease inhibitor cocktail by pipetting the pellet up and down several times. Incubate on ice for 30 minutes.

Every five minutes, vortex for 10 seconds at maximum speed. Centrifuge at 12, 000 x g at four degrees Celsius for 10 minutes. Collect the supernatant and transfer into mini dialysis devices with the maximum volume of 100 microliters per unit for desalting.

Cap the mini dialysis devices and place them in a flotation device. Put the flotation device in a beaker containing 500 milliliters of pre-chilled dialysis buffer. Be careful to avoid direct contact with skin or inhalation of the dialysis buffer, because it contains PMSF.

Incubate at four degrees Celsius with gentle stirring for 30 minutes. After 30 minutes, collect the samples from the corner of the mini dialysis devices and transfer to new 1.5 milliliter microcentrifuge tubes. Centrifuge at 12 thousand times g at four degrees Celsius for 10 minutes.

Aliquot 25 microliters of each supernatant into a 1.5 microliter centrifuge tube and store at minus 80 degrees Celsius. Prepare the protein AG sepharose beads to be used in the immunopreciptation. Pipette 50 microlitera of beads into each of four 1.5 milliliter microcentrifuge tubes and add 500 microliters of DPBS buffer per tube.

Pellet the beads by centrifugation. Discard the supernatant and resuspend the beads in 100 microliters of TBS buffer. Add two microliters of mouse monoclonal anti-ARNT antibody or mouse immunoglobulin G that was prepared by reconstituting 0.7 milligrams of mouse IgG in 500 microliters of TBS buffer.

Take the microcentrifuge tubes containing the beads into a cold room. Place them in a tube rotator and incubate at 10 rpm for two hours. After two hours, pellet the beads by centrifugation and discard the supernatants.

Dilute 200 micrograms of the nuclear protein lysate prepared earlier in 800 microliters of immunoprecipitation buffer. Incubate the lysate with the antibody-coupled beads at four degrees Celsius overnight. On the following day, pellet the beads by centrifugation at 3000 times g for two minutes at four degrees Celsius.

Discard the supernatant. Add one milliliter of ice-cold TBS containing 0.2%NP-40 to each tube and spin down. In this manner, wash the beads three times with ice-cold TBS containing 0.2%NP-40.

Boil the beads in 50 microliters of Laemmli sample buffer at 95 degrees Celsius for five minutes. Centrifuge the beads at 10 thousand times g at four degrees Celsius for five minutes. Collect the supernatant and discard the beads.

The expression levels in subcellular localization of the components of the HIF-1 complex following hypoxia treatment were examined in HEK293A cells. As expected, total HIF-1a levels were upregulated by hypoxia whereas ARNT levels in total cellular lysates were not significantly altered. In addition, hypoxia induced nuclear accumulation of both HIF-1a and ARNT.

Co-immunoprecipitation experiments were performed using nuclear extracts from HEK293A cells exposed to normoxic or hypoxic conditions. As shown here, HIF-1a was co-immunoprecipitated together with ARNT from the nuclear extracts of hypoxic HEK293A cells, indicating that HIF-1a interacts with ARNT under hypoxic conditions. After its development, this technique paved the way for researchers in the field of hypoxia to explore endogenous nuclear protein protein interactions and their hypoxic conditions.