Week 9 Lab: Protein Purification & Mycelium

Fungal Materials Follow-up

The first thing we did was look at our mycelium molds (no pun intended). We are tracking the growth and structural integrity of the fungal networks as they colonize the substrates.

Mycelium Growth Mycelium Growth

Protein Purification: An Introduction

To isolate our protein of interest, we first had to grow the cells and then lyse them using a combination of B-PER (Bacterial Protein Extraction Reagent) and sonication. This process breaks open the cell membranes, resulting in a lysate solution (labeled as Tube A) that contains the total protein content of the cells.

Method 1: Magnetic Bead Protein Purification

This method utilizes functionalized magnetic beads to “grab” tagged proteins out of the lysate.

The Procedure:

  1. Binding: Added magnetic beads to the solution (labeled Tube B).
  2. Separation: Took 500µL of the mWatermelon + mag beads and placed them onto a magnet tray. The beads (now bound to our protein of interest) sat tight against the magnet.
  3. Clearing: Pipetted out the residual volume (excess proteins and buffer).
  4. Washing: Added 500µL of Wash Buffer (containing a small amount of imidazole, 20mM) to wash any untagged, non-specific proteins from the beads. Mixed well and returned to the magnet tray to remove the wash.
  5. Elution: Added 200µL of Elution Buffer (containing a high concentration of imidazole, 500mM) to strip the protein from the beads.
  6. Collection: After the beads settled on the tray, we pipetted the liquid out. This is Solution 4.
  7. Repeat: Repeated the elution step with another 200µL of Elution Buffer to catch any residual fluorescent protein. This resulted in Solution 5, which still contained a significant amount of FPs.

Magnetic Bead Setup Magnetic Bead Setup Separation Process Separation Process Wash Step Wash Step Final Eluted Protein Final Eluted Protein

Method 2: Ni-NTA Spin Column Protein Purification

As an alternative to magnetic beads, we used Ni-NTA (Nickel-Nitrilotriacetic acid) beads in a spin column format, which relies on centrifugal force to move the buffers through the resin.

The Procedure:

  1. Incubation: Combined 200µL of Ni-NTA bead solution with 2mL of lysate. We let this incubate for 30 minutes to allow the His-tagged proteins to bind to the nickel resin.
  2. Binding Spin: Transferred to an eppy tube and spun at 8,000 RPM for 1 minute. We collected and observed the flow-through.
  3. Wash Step: Added 500µL of wash buffer to the column and spun again at 8,000 RPM for 1 minute.
  4. Elution: Added 200µL of Elution buffer and performed a final spin at 8,000 RPM for 1 minute.
  5. Observation: We analyzed the final flow-through to confirm the presence of our purified protein.

Spin Column Loading Spin Column Loading Centrifugation Step Centrifugation Step Column Wash Column Wash Final Purified Sample Final Purified Sample

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