Looking for huntingtin interacting partners – HMGB1 and iVHH4

In my continued bid to find protein molecules which might bind and stabilize the huntingtin (HTT) protein molecule for my structural biology experiments, I have used a technique called pull-down to assess binding of the DNA binding protein HMGB1 as well as the HTT nanobody iVHH4 which I previously described and purified.

In this case, I immobilized HTT mixed with these different protein samples onto FLAG beads to see if they co-purified with the HTT but unfortunately I did not see any binding. iVHH4 should serve as a positive control in these assays as it was previously shown to bind HTT in a similar co-immunoprecipitation assay.

SDS-PAGE of the pull-down assays – bands for iVHH4 and HMGB1 are not seen in the eluted (E) lanes in the control samples or when run with HTT

I have some ideas for why the assay might now have worked as well as how to repeat and improve the assay which I will be testing out this week so fingers crossed I can definitively demonstrate binding, or not as the case may be.

7 thoughts on “Looking for huntingtin interacting partners – HMGB1 and iVHH4

  1. Rachael Jane Harding, What Open Science Tools are you currently using? Examples: Open Access/Open Research Repositories, Preprints, Open Citations, Open Annotations (Hypothes.is), Electronic Lab Notebooks, etc. For example, Have you considered using A.I.? Since Artificial Intelligence easily recognizes shapes and patterns, why not see if you can apply Artificial Intelligence (general purpose learning algorithm(s) /reinforcement learning), i.e. Google AphaGo/Deep Mind, so it will learn to explore and identify protein molecules that will bind/fit and stabilize the Huntington (HTT) protein molecule? As a result, you might even find protein molecule variants to consider that will work as well. You might consider combining your genome sequence data and genetic sequence data available at GenBank http://www.ncbi.nim.nih.gov/genbank . Has it been done before, not yet. Think: The impossible is I’m possible. Why not suggest it as a project challenge for the Deep Mind team to consider to work on? If they accept your challenge, and succeed, it would help other researchers to test other A.I. discovered/promising protein molecules in their labs as well. It doesn’t hurt to ask if it can be done? Use the Scooby-Doo motivator. Think outside the Lab. Use your imagination and curiosity to create new paradigm shifts in Open Data Research and Medicine. Other Open Science Tools you might consider using…
    Are you using the Semantic Web/IBM Watson to parse and read scientific literature in your field? Open Pediatrics at http://www.OpenPediatrics.org is testing it. Are your lab protocols/reagents that you use in your lab found/listed at protocols.io http://www.protocols.io and Addgene http://www.addgene.org? Are you using the Open Science Framework http://osf.io for your workflows ? What electronic lab notebooks (ELN) do you use? Examples….Jupyter http://www.jupyter.org ; SciNote http://scinote.net ; or maybe the European Union’s 2020 Open Data/Fair Use Data Cloud model soon to be released? Have you recently tried Digital Science Dimensions at https://www.dimensions.ai for literature review? O.K. Time for me to stop…tmi (too much information) for you to digest. Enjoy πŸ™‚

    1. Thanks so much for taking the time to send me all of these suggestions, it is very kind of you. I have added them to my resource list – its always helpful to see what other folk are doing. I hope you keep following my open notebook as well as those of the other SGC scientists πŸ™‚

  2. Rachael Jane Harding, Will your Open Lab Notebook include an open access reading section? For example, available open access books about Huntington’s at IntechOpen http//www.intechopen.com/search?q=huntington’s+disease Enjoy πŸ™‚

  3. Rachael Jane Harding, Going off in a tangent here. Are you familiar with nitrate poisoning in animals? The nitrate poisoning happens when their feed is tainted with nitrogen from agricultural fertilizers, such as in alfalfa cubes. It’s a slow, gradual accumulative process that leads to nitrogen bubbles in their blood leading to impaired CNS functions. I have seen it my friend’s horse Baron that my Dad (Veterinarian) treated with 10% methylene blue in solution(Ringer’s/Glucose)/. The methylene blue fixates the free nitrogen in the blood and it’s like a miraculous recovery, seeing a horse with human-like dementia recovering fully within twenty minutes as nothing had happened. Oh, you have to buy methylene blue crystals at a chemical supply house to make the 10% methylene blue solution. It’s rather funny when you consider that the methylene blue commonly used in microbiology for slides is the antidote for nitrate poisoning in animals. Also note, horses have more erythrocytes than humans. Enjoy πŸ™‚

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