8th June 2016 – Day 13

Wednesday:

29˚C in the lab today, we had everything open, windows, doors, everything and it was still too hot. The university was having some form of a sports day and we could hear all the cheering coming in through the windows. We all kept getting a fright as the horn sounded throughout the day, creating a nice atmosphere and keeping us on our toes despite the heat of the day.  (We all looked for an excuse to go down to the phd labs, or the room at the back where we get our ice from, the rooms with the air conditioning .... just so we could cool down a bit.) 

Today we finished off the third protein purification, taking the same samples as we went along to run on a gel. 

We concentrated the third eluted protein the same way we did the Sufu and it was applied to the GF column.

We really knew what we were doing today as we have done it so many times this week, this made things a little more straight forward which was a good thing because of the heat.

7th June 2016 – Day 12

Tuesday:

We analysed the Sufu fractions from the filtration column on a gel. We then pooled the fractions from the GF column (concentrated and quantitated, aliquoted and flash frozen).

The column was washed with water and ethanol and we had to change the size of the column for the second protein which is much smaller in size.

The Gel Filtration Column

The Histag purification was completed with the second protein and samples were taken at each stage and run on a gel.

Looking at the gels, most of the proteins for both were found to be insoluble and we decided to do instead of do can you say run the cultures again but this time incubate them at 18˚C instead of 37˚C overnight. So we began again, making fresh cultures for all and a new broth for the giant culture flasks. We decided to go ahead with the purification of the third protein which we did and incubated overnight again with rotation.

I keep thinking its Thursday, we have done so much in the last 2 days that I feel like it should be the end of the week. I guess the heat doesn’t help, the labs have been incredibly hot (the weather report lied, they said it would rain, it never did, we need just a little rain to cool us down).

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).

3rd June 2016 – Day 10

Friday:

Buffer day!! We made all of the buffers and solutions we are going to need for next week (in preparation for what we are planning to be a very busy week.)

1 litre of Buffer A – wash buffer

1 litre of Buffer B – elution buffer

One would think these buffers would be easy to make by following the instructions, but for some reason we all kept getting confused, it took us 3 attempts to get it right (at first we added the concentrations, instead of the volumes and then we put the opposite volumes of Imidazole in to each….really its like we were all having a momentary lapse of reason......they are only buffers). These were both pH’d to pH 7.6 using concentrated HCL. The pH meter had to be calibrated beforehand between pH 10 and 7. These will be needed for the pull down assays next week.

2 litres of gel filtration buffer

1 litre H20

1 litre 20% ethanol (made from 100%)

These last 3 solutions were filtered and degassed, it took a while but it needed to be done for the gel filtration column, it can’t have any bubbles (although who doesn’t love bubbles).

The column is stored in ethanol to stop bacterial growth, so it is washed with water and gel filtration buffer (this is to prepare it for use next week).

It's been such a busy two weeks, but I have really enjoyed every minute. My poor feet are looking forward to some downtime or should I say ''up time'' and a bit of rest this weekend, but I am really excited to see what next week holds. 

Day 9 – Blot Results!!

2nd June 2016 – Day 9

Thursday:

Western blot continued… after the membrane was washed with blocking buffer overnight, it was washed twice with 1x TBS for 10 minutes each. During this time more blocking buffer was made, 10ml for each membrane with 10ul of the corresponding antibody. It was rocked for an hour in the primary antibody. While the membranes incubated in antibody we made some TBS/Tween/Triton buffer after which each membrane was washed twice with this buffer and then twice with TBS/Tween/Triton. Each membrane was then incubated again for an hour in secondary antibody. The same antibody can be used for each membrane as these are not protein specific but can bind to any antibody. Each membrane was washed twice again with TBS/Tween/Triton buffer and then twice again with TBS buffer. Once we had completed this we incubated membranes for 2 minutes each in chemiluminescent reagent and detected the proteins using the pixi (fancy machine down stairs, my favourite).

Pixi (the fancy machine)

We had stuck the gels into de-stain and then photographed each one (this was just to make sure that all the proteins had been transferred to the membrane, a double check if you will).

The cultures from the conical flasks (made yesterday) were harvested by centrifuging for 20 minutes at 8000rpm and 4˚C. The pellets were fished out and frozen in the -80˚C freezer.

1 conical flask was prepared with LB broth and autoclaved in preparation for the streak plate culture, Sufu, then a single colony was taken from the streak plate and was cultured, induced with 0.4mM IPTG and incubated at 18˚C overnight.

I've known the theory of how to carry out a Western blot, so to actually do one start to finish in the lab was very exciting (Go me! Whoop!)

1st June 2016 – Day 8

Wednesday:

Today was the first day of many western blots! (A process of detecting proteins using antibodies). We placed all 4 gels (that we made yesterday) into the tanks, and loaded all of the samples onto each gel. These were run at 100V and 180V, for 1 hour and then 2 hours. 

I made 3% blocking buffer in 1x TBS (this was just milk powder and water, first the potatoes now milk, what next….?.... I am hoping for something cake related). 

Some of the buffers and reagents needed for a WB.

Once the gel had finished running, we needed to transfer the proteins to a nitrocellulose membrane. (The order for transferring: mesh, filter, membrane, gel, filter, mesh, each mesh was surrounded by a plastic cassette). Each was dipped in 1x transfer buffer. The tank was filled with transfer buffer and the assembled cassettes for each were placed in the tank (it holds 4 and therefore each one fits in nice and snug). The transfer tanks were run at 100V, 350mA for 60 minutes. The current didn’t get high enough and there was no visible ladder on the membrane as the frozen ice packs were placed in the wrong position so we had to remove these. We took the cassette apart and soaked the sponge pads and membranes for 15 minutes after which we ran the transfer for a further hour. The membranes were removed, washed with TBS twice for 10 minutes and left to soak in blocking buffer overnight.

After the proteins were transferred from the gels to the nitrocellulose membrane, we put all of the gels in Coomassie blue stain (so we could look at it later).

Gels put into stain.

The overnight cultures we did yesterday were transferred  to a (100ml) conical flask, with 100ul KAN. I showed the volunteers how to make more IPTG (100mM) and how to filter sterilise, by teaching this process it helped solidify it for me. After some time, the OD readings were taken, at 0.573 OD reading they were transferred to the large flasks (you know…. the really big ones). 20ml of culture into the giant flask with the corresponding antibody. It was then incubated for 2 hours. After the 2 hours had passed the OD was measured. It was not high enough and so it was incubated for another hour and 30 minutes, after which the OD was measured at 0.521. The IPTG was added to each culture and incubated overnight.

The western blot was trial and error and we didn’t expect that it would work straight away. We are mostly preparing and practising the protocol for the next few weeks when we really need it. We had to juggle both culturing and western blots throughout the day….(quite challenging)….but what a great day (happy dance).

31st May 2016 – Day 7

Tuesday:

Two new volunteers in the lab today, and Amy charged me with showing them the ropes and overseeing things. I know them both well from class so at least we are all familiar (but one thing I have learnt today is I definitely like being in charge!).

We didn’t do anything new today, it was all similar methods to last week, but it allowed me to build some confidence as I knew what I was doing and what the processes were. I showed both volunteers how to make SDS-PAGE gels, we made 4 and prepared the 10% APS on our own. 

We got a chance to photograph the gel that I ran on Friday (the one that had been de-staining over the weekend). This worked really well (but note to self don’t make gels symmetrical with the marker in the middle, there is no way to tell which way round it should be, d’oh!).

The gel dried out too much and it caused it to become brittle.

We made more buffers, ones we will be requiring for the western blot at a later date. We made a litre of Transfer buffer and a litre of fresh 10x TBS buffer, which we had to pH using a probe. I never realised how hard this would be, it took forever to get the buffer to the correct pH (7.6) to be exact. The probe was calibrated at the start and washed well between each solution.

We did a streak plate of SUFU using the glycerol stock, we will be using this to make up larger liquid cultures in the future. We made two small liquid cultures of 5ml LB each, 2 using KAN and these were incubated overnight. We prepared 2m 100ml liquid cultures in conical flasks and autoclaved them in preparation for tomorrow.

It was a very productive day, we got through loads of work and it definitely helps having the extra pairs of hands.

30th May 2016 – Day 6

Monday:
Bank holiday extra day at home with my feet up and the family for company! Bonus!

27th May 2016 – Day 5

Last day of the week, cannot believe how quickly that went! There wasn’t too much left to do as Amy wants to leave the big stuff for next week and I am told we will be getting more help, this will definitely be useful always nice to have an extra pair of hands.

The flasks were removed from the overnight incubators, 1ml samples were taken from each. The samples were spun, the supernatant was removed and pellets were frozen (I will need this in a few weeks time). The samples I took yesterday as well as the ones I took today were prepared for another SDS-PAGE gel, they were added to 4x loading dye and pulled through a needle (this was to break open the cells). Gel (which I made on Tuesday and stored in the fridge) was prepared in the tank while the samples were heated at >90˚C for 5 minutes. 20ul of each sample was loaded onto the gel along with 10ul of marker (this is used to tell the size of each separated protein), this was the same process as Tuesday. The gel was stained and then left to de-stain over the 3 day weekend. We will have a look on Tuesday and see if it has worked.

I also used the chilled centrifuge to pellet the reaming cultures in the 50ml falcon tubes, the pellets were then frozen.

The chilled centrifuge: used to pellet the cultures (Clair the technician said it was older than me).

Hard to believe 1st week is finished, its been so exciting. I have loved every minute of getting to know everyone and learn where everything is, whilst getting a taster of what it would actually be like to work in a lab. I loved even loved the hard bits (just means I am being challenged).