The overall goal of this Affinity Capture Protocol is to isolate endogenous protein complexes from mammalian cells. This method can help answer key questions in cell and molecular biology, such as which proteins form together into physically-linked functional modules. The main advantage of this technique is that numerous physical interactions associated with the target protein of interest may be observed simultaneously.
Grow one to eight grams of cells as described in the text protocol. Pour off the growth medium into a large beaker. Then, place the culture dish on ice in a large rectangular icepan.
Add 20 milliliters of ice-cold 1x phosphate-buffered saline, or PBS, to the culture dish and release the cells from the dish using a large cell scraper. Transfer the cells to a 50 milliliter tube pre-chilled on ice. Add an additional 10 milliliters of ice-cold 1x PBS to the same dish.
Collect the remaining cells and transfer them to the 50 milliliter tube. Repeat this process for each dish. Cell suspensions from different dishes may be combined to reduce the sample number and plastic waste.
Next, centrifuge the samples for five minutes at 1, 000 x g in four degrees Celsius. Following centrifugation, carefully pour off the supernatant. Resuspend each pellet in ten milliliters of ice-cold 1x PBS.
Consolidate the resuspended pellets at up to five per 50 milliliter tube. Wash the empty tubes with 10 milliliters of PBS. Add the solution to the consolidated samples and centrifuge as before.
After removing the supernatant, resuspend the pellet in 10 milliliters of ice-cold 1x PBS. Remove the plunger from a 20 milliliter syringe and set it aside. Cap the nozzle of the syringe, place the syringe inside a 50 milliliter tube, and transfer the cell suspension there.
Centrifuge for five minutes at 1000 x g, four degrees Celsius. Aspirate the supernatant with a fine tipped pipette attached to a vacuum trap system until the top layer of cells begins to be sucked up. This results in a wet cell pellet.
Uncap the syringe, insert the plunger and drip the cells directly into a large plastic beaker filled with liquid nitrogen held in a liquid nitrogen bath in a styrofoam box. Forcibly plunge the remaining cells from the syringe. Next, pour the frozen cells into 50 milliliter tubes.
Loosely cap the tubes to allow excess liquid nitrogen to evaporate. Hold them overnight at 80 degrees Celsius. Tighten the caps fully the next day.
The frozen cells may be stored in this way at 80 degrees Celsius until cryomilling. Remove the cell beads from the 80 degree Celsius freezer and place them in a 50 milliliter tube holder in a liquid nitrogen bath. Pre-cool a 50 milliliter jar, two 20 millimeter balls, and the jar lid in a clean rectangular ice bucket containing liquid nitrogen.
Also, pre-cool a PTFE insulator. Use either a set of pucks or a sleeve-and-puck. Set the appropriate counterbalance on the mill.
Using forceps, place the two pre-cooled 20 millimeter milling balls and the frozen cells inside the pre-cooled milling jar. Add liquid nitrogen to the jar up to half full, place the lid on the jar and transfer the assembly to the mill. Clamp the assembly in place and perform three cycles of milling, using a program of 400rpm, three minutes, reverse rotation each minute, and no interval break.
It is critical to hear the balls rattling during milling, this indicates that the balls are colliding and pulverizing the material. Failure to hear this sound means that milling is not occurring. Upon completion of three milling cycles, move the jar back to liquid nitrogen and let it rest momentarily to cool.
Carefully remove the lid. Remove the balls using forceps and transfer the powder to a pre-cooled 50 milliliter tube using a pre-cooled spatula. Note that adding liquid nitrogen to the jar can help dislodge powder that is caked onto the balls.
For cell extract preparation, weigh out 100 milligrams of cell powder into a pre-cooled 1.5 or two milliliter microfuge tube. Tare an analytical balance with the empty microfuge tube. Dispense the cell powder into the tube using a liquid nitrogen cooled spoon or spatula.
Check the mass of the powder dispensed within the tube on the analytical balance before returning the tube to liquid nitrogen. Open the tube with cell powder and let it stand at room temperature for one minute to release pressure within the tube and prevent the immediate freezing of the extraction buffer when added to the powder. Add 400 microliters of extractant, supplemented with protease inhibitors to the tube.
Vortex the tube briefly and then hold on ice. Use a micro tip ultrasonicator to give the sample a brief low-energy pulse and disperse any aggregates. Clarify the extract by centrifugation.
Finally, remove the supernatant and proceed to the affinity capture. To pre-wash the affinity medium, place the tubes containing antibody-coupled paramagnetic beads on the magnet. The beads will accumulate on the side of the tube within seconds, permitting the storage solution to be removed.
Then, add 500 microliters of extraction buffer to the beads. Briefly vortex at medium speed to mix. Pluse spin the tubes in a mini centrifuge to collect all contents to the bottom of the tube.
Then place the tubes on the magnet and aspirate the solution. Initiate the affinity capture by transferring the clarified cell extract to the tubes containing pre-washed affinity medium and vortex briefly. Incubate the tubes for 30 minutes at four degrees Celsius with continuous gentle mixing on a rotator wheel.
The beads should remain suspended throughout the incubation. Aspirate the supernatant and wash the beads three times with one milliliter of cold extraction buffer. To elute the proteins, add SDS page sample buffer without reducing agent, to mitigate the release of antibody chains from the beads.
Incubate for five to ten minutes at room temperature with agitation. Collect and save the supernatant, then subject the samples to SDS page, followed by protein staining using standard techniques. Individual protein bands may be excised for identification, or the entire sample may be characterized in a single analysis by electrophoresing the sample only briefly.
The material comprising the insoluble medium used for the affinity capture can affect the recovered proteome. Shown here are magnetic beads, iron impregnated agarose, and traditional agarose. Each medium was coupled to anti-FLAG M2 antibodies, and used to capture FLAG-tagged bacterial alkaline phosphatase spiked into extracts produced from cryomilled HEK 293 cells.
Micron-scale magnetic beads showed the cleanest profile, indicating a relatively low level of non-specific protein adsorption. The method of cell lysis can also affect the recovered proteome. Here, an endogenous protein complex was subjected to affinity capture from cryomilled, or sonicated cell extracts.
Fewer high-mass contaminants were observed when the cell extract was produced from cryomilled cell powder. Even though magnetic media may exhibit lower background than other media, the antibody itself may facilitate the adsorption of false positive contamination, especially in the absence of its target antigen. To determine the background binding contributed by the anti-FLAG magnetic medium when antibody paratopes are, or are not occupied, mock affinity capture experiments were conducted using HEK 293T cell extracts in the presence, or absence, of a 3x FLAG peptide spike-in.
In the absence of its cognate epitope, alpha FLAG medium does capture a detectable level of background proteins that can be competitively eluded with the 3x FLAG peptide and eluded to a greater extent by SDS page sample buffer. A dominant contaminant species was observed at between 37 and 50 kilodaltons. In the presence of a competing epitope, both 3x FLAG and SDS based elutions are comparably free from background protein binding.
While attempting this procedure, it's important to work carefully, rapidly and precisely. Keep track of your procedures and any variations observed, to learn which steps are most critical for your results. Following this procedure, other methods such as mass spectrometry, rate-zonal centrifugation, and electron microscopy can be performed to analyze the composition of the mixture, its level of homogeneity, and the size and shape of constituent macromolecular complexes.
After its development, affinity capture paved the way for researchers in the field of proteomics to explore the compositions of physiological protein complexes in unparalleled detail, which remains an ongoing global research effort. After watching this video, you should have a good understanding of how to prepare mammilian cells for cryomilling, and how to execute affinity capture of a protein of interest.
