Gill Tissue + Sound Waves = Protein Soup

Sonication uses sound waves to agitate the particles of a solution. For our purposes, sonication allows us to lyse cell walls and extract the protein inside. Here’s how we did it!

Once again, our protocol was adapted from Rhonda’s work.

Added 50 mM NH4HCO3 + 6M urea solution to samples

• Removed samples from -80ºC freezer and placed in rack
• Pipetted 500 µL 50 mM NH4HCO3 + 6M urea solution into one sample tube
• Repeated for all samples + 1 blank

Homogenized samples

• An up-and-down motion worked better than a side-to-side motion (pounding > smushing)
• Homogenized as much as possible
• Resulting mixture was cloudy, difficult to see tissue fragments
• Repeated for all samples + 1 blank

Vortexed samples

• 3 pulses on vortex set to speed 10 (maximum speed)
• Repeated for all samples + 1 blank
• Place all samples in dry ice
• NOTE: This was something we didn’t know we needed to do until Emma told us to ice our samples. The samples were off ice for approximately 1.5 hours

After these steps, we took dry ice, wet ice and our samples over to the Genome Sciences building to use their sonicator.

Sonication preparation

• Filled containers with 1000 mL nanopure water each and labelled containers “1” and “2”
• Made an ethanol dry ice bath
• Filled a beaker with ethanol
• Added one piece of dry ice at a time
• Continually added ice over the course of sonication
• Ensured all samples needing sonication were in a wet ice bath
• Cleaned sonicator tip
• Dipped in ethanol for 5 seconds while on
• Dipped in first container of nanopure water (1) for 5 seconds while on
• Dipped in second container of nanopure water (2) for 5 seconds while on
• Wiped down with additional ethanol and a kim wipe

Sonication

• Sonicated one sample
• Placed sonicator tip in one sample centrifuge tube for 10 seconds
• Immediately placed sample in ethanol dry ice bath for 5 seconds
• Moved sample to wet ice bath
• Cleaned sonicator tip using procedure explained above
• Repeated for the rest of the samples sequentially, and the blank
• Repeated sonication procedure for all samples and a blank 3 times total
• Sonication must proceed sequentially (one sample sonicated, then another sonicated)
• Cannot sonicate one sample immediately after it has already been sonicated
• MUST SONICATE REMAINING SAMPLES BEFORE RETURNING TO ONE SAMPLE

We sonicated Laura’s samples first. My samples froze when in dry ice, so we had to thaw them before I could sonicate. While I sonicated my samples, some of them looked “frothy” (think whipped egg whites). Emma assured me that it was normal. The samples that were frothy while sonicated were O15, O37, O55, O77, O119, O127, O142.

Once we finished sonication, we returned back to our lab for the final step. We carried our samples in wet ice.

Transferring sample to a new centrifuge tube

• Vortexed sample
• 3 pulses on vortex set to speed 10 (maximum speed)
• Pipetted 11 µL of the sample into a new centrifuge tube with the label “11 µL”
• ex. 11 µL from “O07 prot” to “O07 11 µL”
• Placed “11 µL” centrifuge tube in wet ice with the rest of sample tubes
• Repeated with all samples and blank

And that’s it for sonication! I placed all of my sample tubes (prot and 11 µL) in the -80ºC freezer. Tomorrow, we move on to BCA Analysis.