6th June 2016 – Day 11

Monday:
One volunteer down today, gone for a whole week… the lucky person has gone on holiday!!

The rest of us prepared 50ml Lysis Buffer (it doesn’t help when you forget the protease Inhibitor tablet the first time round!). The Sufu that was grown in the large flasks last week was harvested today (the same way as before, in the chilled centrifuge), the pellet was frozen at -80˚C for an hour and then thawed and resuspended in 16ml lysis buffer.

The -80 freezer, always makes my hand go bright pink!

While we did this we also made 4 gels, we made doubly sure that we added everything, and decided to use the expensive gel equipment from downstairs because we were told that it would reduce the cross contamination between wells and produce more clear results… so we shall see.

Once the pellet had been resuspended, we sonicated the pellet with the large probe (this uses sound waves to break open the cells releasing any proteins inside, it makes a horrible high pitched noise… but it meant we got to wear some very awesome looking bright yellow noise cancelling headphones…we looked very cool). We took a small sample to run on the gel and put the rest of the Sufu sample in the centrifuge, 1700rpm for 20 minutes. The supernatant was filtered off and the pellet was resuspended in TBS buffer (a sample taken from both for the gel).

The sonicator and those cool headphones

We did a histag-purification of the supernatant using nickel beads, the beads were washed well with buffer A and then the supernantant was added and left to incubate with rotation for an hour at 4˚C.

We used this time to take our lunch break and we decided to take it outside, as it was a nice, hot, sunny day.  This turned out to be a bad idea, I had the worst headache in history… for the entire afternoon and my lunch partner ..... well she was convinced she had heat stroke, she was rather pale to be honest. This made the afternoon slow but we soldiered on and still manged to finish everything we needed to.

We carried on with the purification, we poured the incubated Sufu sample into a column, we took all of the necessary steps to collect the flow through, one wash, the elution and then we resuspended the beads in buffer B (elution buffer). We took a sample from each of these and ran them on a gel.

The elute was concentrated using a 50K cutoff concentrator and applied to a gel filtration column. The same process was done to the second protein and it was left to incubate with rotation overnight (we will carry on with the purification tomorrow).