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!)