Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 7.13.08
- Run putatively purified HmgR on Ion-Exchange column to further remove contaminants and run gel to assay purity.
I took the HmgR that I purified on Friday and ran on a Q-Sepharose ion-exchange column to further purify it. The protein looks more pure. I dialyzed over night and will concentrate the product tomorrow. After concentrating and determining the concentration of the final product, I'll start writing in the work log entitled, "HmgR Binding Assay," as my purification work will hopefully be done.
- Concentrate the doubly purified HmgR, add glycerol, and store at -20. When I return next week, I'll do a Bradford to determine the actual concentration.
I concentrated my protein sample to ~400 μl from 15 ml of dialyzed HmgR. To this 400 μl I added 400 μl 100% glycerol, mixed well (without forming bubbles) and stored at -20. When I return next week, I'll determine the concentration of protein. I also need to run both the purified HmgR and p'So's GpxR on a gel to determine the purity. Mayuree said to use ~2-5 μg per lane.