
Year: 2017
Huntingtin sample preparation before the holidays
Before the university shuts down for the holidays, I have been busy in the lab making huntingtin protein samples. I purified wild-type huntingtin with a polyQ stretch of Q23 as well as disease versions of the protein with Q54 and Q145. Whilst Q23 huntingtin protein can be trivially expressed and purified, the longer polyQ length Read More …
Huntingtin nanobody expression and purification (2017/11/24)
Recently, I was kindly provided with nanobody constructs which binds the proline rich region of huntingtin exon 1 (the first 90 amino acids) by Ray Truant (McMaster University). Nanobodies are small antibody like molecules containing just a single Ig domain which have high affinity for a specific protein and ideally a specific part or motif Read More …
Mapping post-translational modifications of huntingtin from insect cells
I have been continuing to characterise the huntingtin protein samples I am generating in the lab. You can read about my first attempts to map post-translational modifications by mass spec here. Previously I found phosphorylation modifications on the huntingtin protein which are located on the same sites as huntingtin protein derived from human cells which Read More …
Making huntingtin nanobodies
Prof Ray Truant kindly shared his nanobody construct with us. This encodes a camelid derived Ig domain which binds huntingtin as he describes in this paper. This may be helpful in stabilizing our huntingtin protein samples to make it easier to work with in our structural experiments. Our cloning team recloned the nanobody DNA sequences Read More …
Post-translational modifications of insect cell derived huntingtin and success with HMGB1
Hi folks! Recently, I have been doing some work to further characterise the huntingtin protein samples I am generating in the lab. My huntingtin protein is made in insect cells and these cells process proteins they make differently to human cells. In particular, I am keen to see whether a chemical modification called phosphorylation is Read More …
Back from vacation and making huntingtin!
Hello labscribbles readers! I am back from a glorious vacation in the Canadian wilderness and have been back in the lab for the past week. I have purified some more huntingtin protein samples (Q23 – wild type and Q54 – disease state) which you can read about here on Zenodo. These samples I will be Read More …
Experiments on huntingtin interacting protein HMGB1
To improve the samples I am making of huntingtin for structural studies, I am trying to find binding partners of the huntingtin protein. Complexes of huntingtin with binding partners may be more stable and amenable to study by structural methods. HMGB1 is one such reported huntingtin interacting protein. I have now tried to make this Read More …
Cryo-EM updates and more!
This past few weeks have been busy with fellowship applications, writing grants and papers so I am rather behind on keeping you updated but there has been some exciting progress: Susan Lea has made fantastic progress processing cryo-EM data of our samples and we now have a 13 A envelope of the protein: https://zenodo.org/record/841824 2. On Read More …
Huntingtin and huntingtin-interacting protein purification 2017/07/21
This past week I have been working hard to make lots of protein for future experiments. I have successfully purified full-length C-terminally tagged huntingtin. Having the FLAG tag on the C-terminal end of the protein seems to reduce the aggregation of the sample – I have now seen this consistently in 2 different purifications. I Read More …
Purification of high mobility group box 1 protein (HMGB1) – a huntingtin interacting protein
To generate better data for cryo-EM analysis of the huntingtin protein structure, more stable and conformationally homogeneous samples may help improve the particle averages. Purifying complexes of huntingtin with proteins which specifically bind huntingtin may promote such sample stability. High mobility group box 1 (HMGB1) is known to bind huntingtin but, to my knowledge, this interaction Read More …