9th June 2016 – Day 14

Thursday:

Today we took the fractions from the GF column and ran them on a gel.

I love this picture, the bands are so clear, there is no contamination between lanes, this is what an SDS-PAGE gel should like… the way each of the proteins have separated by size… I am pleased with how this has come out.

Once we had decided which fraction from the GF column contained our purified proteins, we pooled the fractions (concentrated, quantitated, aliquoted and flash frozen).

Concentrating columns

The new cultures we made of Sufu and the second protein we harvested (same as before), were frozen, thawed and then the second protein was re-suspended in lysis buffer. The protein was purified the same as before and we collected all of the samples and ran them on a gel… I’ve done this so many times now that I am almost an expert.

The elute sample of the second protein was concentrated and stored overnight.

Once that was all finished we prepared the two buffers which we will need for the pull-down assays next week.

500ml of Amylose Binding buffer

200ml of Amylose Elution buffer

These were nice and straight forward buffers to make, very similar to the purification buffers we made last Friday.

It was a very challenging day, but we got there in the end!!