Monday – Reading Papers

In Monday, no lab activities were done as Clark was feeling unwell and thus did not come into the lab. I spent the day in the office reading papers about the virus we are trying to identify the structure and function of as well as the most similar known virus. I also read into protein affinity tags, nickel column affinity purification, and protein denaturation with urea. This information will be detailed in the poster presentation as well as the PowerPoint for the July 9 presentation.

Tuesday – Cell Lysis and Lysate Preparation (Gravity Columns)

The preparation began with setting up an ice bucket and creating an ice-water mixture to maintain the temperature of the samples throughout the procedure. The sonication buffer, containing 8 M urea, was taken from the 4°C fridge in the FPLC room to ensure it remained cold. 30 mL of the sonication buffer was poured into a 50 mL conical tube, to which 10.5 µL of β-mercaptoethanol (BME) and 300 µL of phenylmethylsulfonyl fluoride (PMSF) were added. Vortexing the solution was done to ensure the PMSF dissolved properly and the BME was evenly distributed to prevent protein degradation during sonication.

The sonication buffer was then poured over the cell pellet until it was fully immersed. The sonicator probe was cleaned using DI water and 70% ethanol, and then wiped with kimwipes. This step was essential to prevent contamination and ensure accurate sonication. The probe was then placed into the buffer, positioned right above or near the cell pellet to ensure sufficient cell disruption.

Sonication was performed with the sonicator set to 7 minutes at 70% amplitude, with a pulse on/off cycle of 01/03. This setting was chosen to break up the cell pellet without overheating the sample. The goal was to disrupt the cells thoroughly, which was monitored by checking for any remaining cell chunks. If clumps persisted, the probe was moved closer to those chunks for better disruption.

During sonication, the blue-capped rotor was retrieved from the fridge and placed in the floor centrifuge to pre-cool it to 4°C. This pre-cooling step was done to maintain the integrity of the lysate during high-speed centrifugation. Once the sonication was complete and the cell pellet fully broken down, the lysate was poured into a compatible centrifuge tube, double-checked to ensure that no floating cell chunks remained.

The lysate was balanced with an identical tube containing DI water to ensure the centrifuge operated safely and effectively. This balance was checked carefully due to the high-speed spin of 30,000 x g for 45 minutes at 4°C, which could potentially damage the centrifuge or pose safety risks if the tubes were not properly balanced.

Post-spin, the clarified lysate was carefully decanted into a pre-cooled 50 mL conical tube, ensuring any remaining cell debris was left behind. This clarified lysate was kept on ice to prevent protein degradation. A small volume of sonication buffer, minus the BME and PMSF, was added to the remaining pellet, resuspending it until the solution turned cloudy. This resuspension helped to prepare the pellet for further analysis.

Following the centrifugation step, the clarified lysate was applied to a gravity column pre-packed with nickel beads, which are specifically designed for affinity purification of His-tagged proteins. The column had been pre-equilibrated with a binding buffer containing 8 M urea to ensure the conditions were optimal for binding the His-tagged proteins. The lysate was allowed to flow through the column by gravity, ensuring that His-tagged proteins had ample time to interact with the nickel ions on the beads.

After the lysate had passed through the column, the next step was washing the column to remove any nonspecifically bound proteins and other contaminants. This was done using the same binding buffer but with a low concentration of imidazole (typically around 20-50 mM). The imidazole in the washing buffer helps to displace weakly bound contaminants without eluting the target His-tagged proteins. Several column volumes of the washing buffer were passed through the column to ensure thorough cleaning. Each wash step was collected and monitored to check for the presence of proteins, ensuring that most contaminants were removed. It was essential to perform multiple washes to achieve a high purity of the bound His-tagged proteins while keeping them securely bound to the nickel beads.

Once the washing steps were completed, the column needed to be stored properly until the elution step was carried out the next day. The column was sealed securely to prevent any evaporation or contamination and stored at 4°C.

Wednesday – Elution & SDS-PAGE gels

The elution process began with the preparation of several elution buffers containing varying concentrations of imidazole: 30 mM, 250 mM, 500 mM, and 1 M. Each buffer also included 8 M urea to maintain protein solubility and a buffering agent to ensure the pH remained stable. These buffers were needed for selectively eluting the bound proteins from the nickel beads based on their affinity for the imidazole.

The gravity column was allowed to equilibrate at room temperature for a few minutes before starting the elution process. Sequentially, each elution buffer was added to the column. The 30 mM imidazole buffer was used first, followed by the 250 mM, 500 mM, and finally the 1 M imidazole buffer. For each concentration, a 10 mL was used to ensure thorough elution. The eluate was collected in separate microcentrifuge tubes, labeled according to the imidazole concentration used. Elution allows for the separation of proteins based on their binding strength to the nickel beads.

After completing the elution steps, a final wash with 1 M imidazole was performed to ensure any remaining loosely bound proteins were removed. This fraction was collected in a separate tube. To confirm that no protein remained bound to the nickel beads, the beads were transferred from the column to a microcentrifuge tube and washed with DI water. The wash fraction was collected for analysis, ensuring that the entire protein sample was accounted for.

Following elution, we ran an SDS-PAGE gel. The purpose of the SDS-PAGE gel was to visualize the different protein fractions and confirm the presence of the target capsid protein 9337 in the 250 mM imidazole elution fraction. Each elution fraction, including the pellet, supernatant, flow-through, and the final wash from the beads, was mixed with protein loading dye. This dye contained SDS, which denatures the proteins and gives them a uniform negative charge, ensuring they migrate based on size during electrophoresis. The samples were heated briefly to ensure complete denaturation.

The SDS-PAGE gel was set up in the electrophoresis apparatus, and the wells were loaded as follows: two wells with loading dye as controls, one with the protein marker, and others with the pellet, supernatant, flow-through, each elution fraction (30 mM, 250 mM, 500 mM, 1 M imidazole), the final wash from the beads, and a second protein marker in the final well.

Electrophoresis was conducted by applying an electric current, causing the proteins to migrate through the gel matrix. The protein marker allowed for the determination of the molecular weights of the separated proteins. The specific band corresponding to the target capsid protein 9337 was expected in the 250 mM imidazole elution fraction, indicating successful purification.

After electrophoresis, the gel was stained to visualize the proteins: the dye solution binds to the proteins, aiding with visualization. This was followed by the de-staining step, done to remove excess dye. The resulting bands on the gel represented the proteins in each sample, which allowed for the assessment of purity and identification of the target protein, which should have been 33 kD but showed as 40 on our gel (something we haven’t understood yet, but will research more about)

That marked the end of this week. Happy (early) 4th of July everyone!! I hope we all have a fun, relaxing weekend.