This protocol describes a complete purification procedure for the human calcium binding protein, S100A12, and in particular, for it's different ion-induced oligomers for downstream research implications. With this technique, we are able to maintain high yield of very pure and endotoxin-free protein. Chemical cross-linking and separation on the size exclusion chromatography column is a convincing method to generate homo-oligomeric proteins for further functional assays.
Our technique allows us to mechanistic aspects of S100A12 as pattern recognition receptor ligand and art information to ideally develop new targeted interventions and improve diagnostic assays. In addition to watching this video, we recommend a careful reading of the protocol, as we describe a complex, multi-step procedure. To begin, cut a dialysis tubing into an appropriate length, around 30 centimeters, with additional space for air.
Soak the membrane in deionized water for 15 to 30 minutes to remove glycerol. To reduce the viscosity of the cleared protein solution, dilute the solution with 25 milliliters of AIEX buffer A.Attach the first closure onto the tubing. Load the sample into the membrane and attach the second closure at least one centimeter from the top end of the tubing.
In a cooled room at four degrees Celsius, place the container with AIEX buffer A on a stir plate, add a stir bar, and the membrane filled with protein solution. Adjust the speed to rotate the sample so that there is no interference with the rotating stir bar. Dialyze for 12 to 24 hours at four degrees Celsius then replace the dialyzed buffer with free pre-cooled buffer A and continue for at least four additional hours.
Transfer the dialyzed protein solution from the tubing into a 50 milliliter tube through a 0.45 micron filter unit. Start the FPLC with general maintenance. Connect column buffers AIEX A and AIEX B to buffer valves and anion exchange resin containing column to the column ports of the FPLC.
Adjust the general chromatographic parameters accordingly. Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A.Subsequently, load the sample onto the column and elute the proteins with a linear gradient from 0%to 100%high salt buffer. Collect two milliliter fractions during elution.
Analyze the eluted fractions after finishing the run. For this, load 10 microliters of each fraction on a Coomassie stained 15%SDS page and identify fractions containing S100A12. Pool these S100A12 containing fractions for dialysis into TRIS-buffered saline.
To continue the protein purification, after the protein dialysis, add a calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns. Equilibrate HIC buffers and samples to four degrees Celsius.
Next, connect column buffers HIC A and B and the column to the system and adjust the parameters. Start the method to equilibrate the column with one to two column volumes of buffer HIC A and load the sample and the wash unbound sample block with a UV signal reaches baseline level again. Then start elution with a calcium chelator containing buffer EDTA.
Collect two milliliters of peak fractions during elution. After analyzing 10 microliters of each fraction on a Coomassie stained 15%SDS page identify pure S100A12 fractions. Pool these fractions and dialyze against HBS.
First, load 15 milliliters of sample onto a 50 kilodalton centrifugal filter unit. Centrifuge at 3200 times g at 10 degrees Celsius for approximately 10 minutes. Transfer the filtrated flow-through into a fresh vessel, on ice.
Now, use one milliliter of HBS to rinse the filter membrane and for dilution of the concentrated solution in the filter tip. Centrifuge again to recover as much protein as possible in the flow-through. To filter the solution, repeat the sample loading and centrifugation as often as necessary.
Next, to concentrate the S100A12 containing flow-through, load it into a three kilodalton centrifugal filter and centrifuge at 3200 times g, 10 degrees Celsius for approximately 30 minutes. Now the volume is reduced to 1/5 up to 1/10 of the initial loading volume. Transfer the concentrated solution to a new tube.
Repeat the concentration step as often as necessary. After chemical cross-linking, according to the manuscript, perform size exclusion chromatography. Equilibrate the column in HBS, load the sample, and collect peak fractions of one to two milliliters during the run.
After analyzing these fractions on a four to 20%gradient SDS page, pool fractions with major bands of the desired protein complex. Then concentrate the solutions as done previously. To isolate monocytes from human buffy coats by density gradient centrifugation, first equilibrate the separation solution to room temperature.
Transfer 20 milliliters into 15 milliliter centrifuge tubes, two tubes per buffy coat. Dilute the blood from the human buffy coat with HBSS to a total volume of 60 milliliters and layer 30 milliliters of this mixture carefully on top of the separation medium. Centrifuge at 550 times g for 35 minutes at room temperature.
Switch off the brake of the centrifuge. After centrifugation, the mononuclear peripheral blood cells are located directly on top of the separation medium. With a pipette, transfer these cells into a fresh, 50 milliliter centrifuge tube.
Fill the tube with HBSS to 50 milliliters and centrifuge at 170 times g for 10 minutes. Aspirate the supernatant and resuspend the cell pellet in a small volume of HBSS by pipetting. Then, fill the tube up to 50 milliliters and centrifuge at 290 times g for 10 minutes.
Aspirate the supernatant again. Resuspend the cells in 50 milliliters of HBSS and centrifuge at 170 times g for 10 minutes. Aspirate the supernatant and resuspend the cell pellet in a small volume of one milliliter of separation buffer, count the cells, and add buffer to a concentration of five times 10 to the seven cells per milliliter.
For monocyte isolation from mononuclear peripheral blood cells, use a magnetic negative cell isolation kit and follow the manufacturer's protocol. Use an automated cell counter to count the monocytes and resuspend in monocyte medium to a concentration of two times 10 to the six cells per milliliter. To culture monocytes, lay out a cell culture dish with a hydrophobic gas permeable film suitable for suspension cells.
Place the plates under UV light for approximately 30 minutes for sterilization. Transfer 15 to 25 milliliters of the cell suspension to these culture plates and let them rest overnight at 37 degrees Celsius and 5%carbon dioxide. Following pre-purification on the AIEX column and subsequent calcium dependent HIC, highly pure protein was obtained.
As an additional control, human monocytes were stimulated with LPS and produced wild-type protein. Protein exposure to different ions results in arrangement of different S100A12 oligomers. Increasing zinc concentrations induce arrangement of S100A12 into tetramers and hexamers upon separation on four to 20%Coomassie stained SDS page.
When an excess of ions was applied prior to cross-linking, it induced a pronounced shift of the oligomer equilibrium. S100A12 was cross-linked in the presence of either 25 millimolar calcium or 25 millimolar calcium and one millimolar zinc. After cross-linking in HBS buffer with 25 millimolar calcium chloride and one millimolar zinc chloride, hexamer and tetramer were separated.
Tetramer and dimer were separated in HBS buffer with 25 millimolar calcium chloride. An example of pooled and concentrated oligomers after separation on a Coomassie stained four to 15%gradient SDS page shows dimer, tetramer, and hexamer. Monocyte stimulation with hexameric S100A12 resulted in pronounced TNF alpha release.
After removal of endotoxin, it is important not to reintroduce contamination into the samples during subsequent generation and purification of the oligomers. Downstream analysis of any process that relates to S100A12 oligomerization is conceivable. Similarly, amino acids and ion conditions that affect the oligomerization process itself can be analyzed.
We're currently using S100A12 oligomers separated according to the described protocol to develop specific peptide ligands and antibodies to block the protein's signaling through TLF4 and to develop and improve diagnostic assays.
