This was the first week back after the storm! Since the ultracentrifugation and gradient method failed to produce significant amounts of protein, we have recently been trying a newer method of protein purification: columns and FPLC.

Monday

Today, I ran gravity columns. The previously centrifuged pellet was in the -80 C freezer. I then added our lysis buffer, 8 M urea with BME and PMSF, and sonicated it to resuspend. Once the pellet was resuspended with no floating particles (completely homogeneous), I centrifuged it on the floor model centrifuge to attain the clarified lysate. After spinning it for 45 minutes, I poured the supernatant into a different tube and resuspended the pellet in 1 mL of lysis buffer (without BME and PMSF) for sampling. I took samples of both the pellet and supernatant (60 microliters of sample and 20 microliters of 4X loading dye for the SDS-PAGE gel to be run later). Once samples were taken, I set up the gravity column and pipetted 4 mL of beads into the column. I equilibrated it with water and our 8 M urea buffer–this is a very time-consuming process involving watching the liquid drip into a separate container and pass through the nickel beads. When the nickel beads were equilibrated, I added the supernatant/clarified lysate acquired from the previous centrifugation and stored it in a fridge.

A brief explanation on gravity columns: Column chromatography functions on the principle that solutes of the solution will be adsorbed with the help of a stationary phase and later separated into discrete components. Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. We spent today washing and planned to elute tomorrow (Tuesday).

Tuesday

Today we eluted the proteins from the supernatant. I started off by collecting flow-through, which was done by opening the valve of the gravity column and allowing the supernatant from yesterday to pass through the nickel beads. The principle is that, upon allowing the supernatant to flow over the beads, the protein with a His-tag will bind to the nickel beads in the column. After this, varying concentrations of imidazole are poured over the beads and collected in various tubes. Clark mentioned that, historically, he has seen success with 250 mM imidazole. Before doing this step, I first had to make solutions with the concentrations of imidazole we would be testing: 30 mM, 250 mM, 500 mM, and 1 M. This was done using 1 M imidazole and 8 M urea (the same buffer we used for lysis). The amounts of each we would use were calculated based on the equation M1V1 = M2V2. We needed 50 mL of 30 mM, 10 mL of 250 mM, 10 mL of 500 mM, and 10 mL of 1 M. Once these solutions of imidazole and urea were made, they were stored in 50 mL conical tubes on ice. The 30 mM was allowed to flow through the beads, but we weren’t expecting much protein to flow with the 30 mM into the elution tube. Following this, the same steps were repeated for 250 mM, 500 mM, and 1 M. This was a very time-taking process as you need to allow the liquid to slowly flow through the small opening on the bottom of the column.

The columns look similar to the image above. We collected samples of the flow-through, 30 mM, 250 mM, 500 mM, 1 M, and beads using the same 60 microliter and 20 microliter ratio I described for Monday. These samples were placed in the freezer, marking the conclusion of today.

Wednesday

Today, we ran SDS-PAGE gels (3 total) and a western blot. My day started by running 2 SDS-PAGE gels (Clark ran the third one). To do this, I removed the samples from the freezer and placed them into the heating block at 98 C. I briefly ventilated the tubes after 2 minutes and allowed them to thaw for 8 more minutes. I also removed the molecular ladder/marker from the freezer and allowed to thaw on the bench. Following this, I removed the gels from the fridge and set up the electrophoresis apparatus. I then loaded the samples into the gel (the two gels contained the same samples, one was meant to transfer onto a western blot, thus we duplicated it). Once the samples, marker, and loading dye were loaded into the gel, I set it to run at 100 V for 15 minutes until the samples hit the separating gel. Next, I set it to 180 V for 45 minutes until the samples run off the gel. When the timer was up, I removed one gel from the apparatus, separated the glass to extract the gel, and stained and de-stained it. It was stained by pouring staining buffer into an empty pipette tip box (repurposed for staining) and microwaving it with the gel for 1 minute. This was placed in the fume hood to vent and later de-stained with clear, de-staining buffer on the rocker. The other gel was used for a western blot (unsuccessful because we did not have the right kind of blot paper). The standard western blot procedure was followed: a “gel-blot sandwich” was created, surrounded by pads and sponges. This was put into the western blot apparatus with transfer buffer and then left to transfer in the fridge at 100 V for 1 hour. During this time there was a fire drill. After the fire drill, when we returned to labs, we found that the contents of the gel did not transfer onto the blot as we did not see the marker on the blot. However, we know there were volts running through the system, so we are hypothesizing that the reason this did not work is because we ran out of the right kind of PVDF paper (so we used alternate PVDF paper). Our gel, which was still on the rocker, was very clear, which is a positive result– it means we removed most of the irrelevant noise surrounding the protein and have isolated the desired protein to a certain extent. This marked the end of Wednesday.

Thursday

Since we do not have PVDF paper, Clark thought it would be interesting if we did a protein refolding experiment. This was just to learn how to run dialysis rather than to get results, as we do not know how successful our purification was due to the lack of PVDF paper. Thus, this was meant to be a learning experience. Today began by creating two different dialysis buffers (as we wanted to test two). Clark made one (with MOPS) that is light sensitive and thus had to be done covered by aluminum foil to maintain a dark environment. I made the other one with Tris, a commonly used compound in buffers. I added NaCl, Tris, 10% glycerol, and water to a 100 mL beaker and used the magnetic stir plate to mix the solution. Then, I filtered it using a vacuum filtering apparatus and stored it in a clean 1 L bottle in the fridge until use. Following this, we took a break/ate lunch and then began the dialysis experiment, which would happen overnight. We took two 100 mL beakers and filled them with our buffers. Then, we let a dialysis membrane/bag soak in the buffer for 5 minutes to soften. Once it had softened, we filled it with 5 mL of the purified protein from Tuesday and clamped both sides of it closed to make it turgent. Following this, we let it sit in the environmental cold room overnight, stirring on a magnetic stir-plate.

Friday

Today was day 2, the final day, of the protein refolding experiment! We were only able to run FPLC for one of the samples (the one in MOPS buffer). We did this by calibrating and equilibrating the FPLC machine. Then, we filled a carousel as per usual and ran the machine (a 2 hour long process). We did not find significant peaks, meaning that there was minimal protein present, which was expected as we noticed most of our protein precipitated (fell out of solution) sitting in the environmental cold room overnight. This was not the only part of today, however. I also had the opportunity to speak with someone who is outside the field of structural biology: they are in synthetic biology. Their name is Malyn and we got coffee altogether with Clark and discussed their project regarding microbes in soil and horizontal gene transfer. I found it exciting to explore various avenues in synthetic biology as well. It’s just a reminder that there are endless directions you can take biology! This marked the end of today.

This week, I did many new things, especially a protein refolding experiment: something I had been wanting to do for a while. We will be running the second dialysis sample through FPLC on Monday; the sample will not be ruined by Monday.