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).