The overall goal of this procedure is to efficiently isolate protein complexes from mammalian cells. This is accomplished by first transecting the cells with a halo tag fusion construct to express the protein of interest. The second step is to slice the cells and covalently capture the protein complexes on resin via halo tag.
Next, the protein complexes on resin are gently washed to remove any non-specific interactions. The final step is to elute the protein complexes from the resin. Ultimately, halo tag pull downs are used to isolate and discover novel protein interactions from mammalian systems.
The methods we will demonstrate here to you today can enable key discoveries in the area of functional proteomics, including identification of novel protein interactions and determination of protein localization inside cells. Performing these procedures today are two of our senior scientists, Jackie Mendez and Alen Beek. First, for each fusion or control, prepare one 15 centimeter dish with 30 milliliters of cells at three to four times 10 to the fifth cells per milliliter, or one to 1.2 times 10 to the seven cells total per construct.
Incubate the cells for 18 to 24 hours at 37 degrees Celsius and 5%CO2. Following incubation, transfect the construct with the desired transfection reagent. After 24 to 48 hours, post transfection, remove the media and gently wash the cell layer with 20 to 25 milliliters of ice cold PBS remove the PBS wash and add 25 to 30 milliliters of four degrees Celsius chilled PBS.
Following this, gently scrape the cells off the dish with a cell scraper. Once the cells have been collected into conical tubes, centrifuge the samples for five to 10 minutes at 2000 times G and four degrees Celsius. After discarding the supernatant, place the cell pellets at minus 80 degrees Celsius for a minimum of 30 minutes or a maximum of six months.
For each fusion or control sample, prepare 18 milliliters of resin equilibration wash buffer. Gently mix the resin by inverting the vial to obtain a uniform suspension. For each pull down experiment dispense 200 microliters of resin into a 1.5 milliliter micro centrifuge tube.
Centrifuge the sample for one minute at 800 times G.Following centrifugation. Carefully remove the supernatant without disturbing the resin at the bottom of the tube. Once the supernatant has been discarded, add 800 microliters of resin equilibration wash buffer to the sample and mixed thoroughly by inverting the tube several times.After.
Centrifuging the sample for two minutes at 800 times. G.Carefully remove and discard the supernatant. Repeat the previous steps two more times for a total of three washes.
After thawing the cell pellets resus, suspend them in 300 microliters of previously prepared mammalian lysis. Buffer by pipetting up and down. Then transfer to a new micro centrifuge tube and add six microliters of 50 x protease inhibitor cocktail.
Following this, add three microliters of RQ one DNAs and invert the sample for 10 minutes. At room temperature ly the cells by passing the sample five to 10 times through a 25 or 27 gauge syringe needle. Once the sample has been centrifuged at 14, 000 times, G for five minutes at four degrees Celsius, transfer the clear lysate to a new tube and place it on ice.
Next, add an additional 700 microliters of previously prepared one XTBS buffer to the clear lysate and mix well by pipetting up and down. Remove the final washes from the previously prepared equilibrated resin tubes without disturbing the resin at the bottom of each tube. Then add one milliliter of the diluted lysate to each tube.
Incubate the samples with mixing on a tube rotator for 15 minutes at 22 degrees Celsius. Following this centrifuge, the resin tubes for two minutes at 800 times G.After discarding the supernatant, add one milliliter of resin equilibration wash buffer and mix thoroughly by inverting each resin tube by hand several times following centrifugation of the resin tubes for two minutes at 800 times. G.Discard the washes once the previous and washing steps have been repeated three times.
Add one milliliter of resin equilibration wash buffer to the tubes after incubating the samples at 22 degrees Celsius for five minutes with constant rotation, centrifuge the resin tubes for two minutes at 800 times G and discard the washes. At this point, resus suspend the resin from each sample in 50 microliters of SDS elution buffer. Shake the tubes at room temperature with an automatic micro centrifuge tube shaker for 30 minutes.
Following centrifugation for two minutes at 800 times G.Transfer the EITs to fresh tubes for analysis For western blot or silver stain gel load five to 10 microliters of the samples on an SDS denaturing gel for mass spectrometry. Store 40 microliters of each sample at minus 20 degrees Celsius for future analysis. In an eight well chambered cover glass for each fusion protein or control plate, 400 microliters of HELOC cells in their appropriate media in each well at a density of one to two times 10 to the fifth cells per milliliter.
After incubating the cells for 18 to 24 hours at 37 degrees Celsius and 5%CO2, transfect them with the desired transfection reagent following 18 to 24 hours, post transfection dilute TMR ligand one to 200 in the appropriate cellular media. Then add 100 microliters of this solution to each well and gently mix. Next, incubate the transfected cells containing the ligand for 15 minutes at 37 degrees Celsius and 5%CO2.
When finished, aspirate off the media containing the ligand and replace it with 500 microliters of the appropriate media lacking protein fusion tag ligand, which has been pre-warned to 37 degrees Celsius. Once the previous step has been repeated twice for a total of three washes. Place the cells back into the incubator for 30 minutes.
After the 30 minute incubation aspirate off media and replace with 500 microliters of appropriate media, which has been prewarm to 37 degrees Celsius. Following this image, the cells on a microscope using the appropriate acquisition parameters using the protocol halo, BRD four and halo tag control expression is observed. Fusion protein expression can also be detected using Western blots with anti halo tag antibodies or antibodies to the bait protein.
Silver stain gels of biological replicates of Halo BRD four and control pulldowns demonstrate high reproducibility and show proteins that interact with the BRD four protein. The high abundance of components from PTF B CDK nine and Cyclin T as well as the BRD nine protein confirms specific capture of BRD four complexes. The gels showed other proteins identified as potential interactors of BRD four.
As an additional example, complex isolations were performed with the Halo HD one protein. In this case, TEV protease was used to cleave in a linker region between Halo tag and H DAC one releasing significant amounts of the HD one bait protein. As observed.
HD one pulldown samples showed high levels of HD one activity, which was inhibited by the HDA inhibitor saha. To further demonstrate specificity, no HDA inhibition was observed with sirtuin family inhibitor EX 5 27 and no signal was detected using buffer alone. He A cells transfected with Halo BRD four and Halo HDAC one were fluorescently labeled with TMR.
Ligand Imaging showed that both localized to the nucleus and demonstrate that the tag did not alter physiological cellular localization of its fusion partners. So following this procedure, the isolated proteins and their interacting partners can be identified and further analyzed using a variety of analytical methods such as mass spectrometry and western blotting.
