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I used a surface plasmon resonance assay to determine the binding affinities of hit compounds that were identified in 19F NMR spectroscopy screens against the zinc finger ubiquitin binding domain of USP5. To see how these assays work, check out some of my previous posts! You can see details of the SPR experiment on Zenodo. Read More …
Hello again! As a follow up to my previous post,I’ve continued the search for an antibody to detect EZH1. I’m happy to report that I finally found one. I was able to detect a band in HEK293 cells using the Cell Signaling Antibody. Using knockdowns targeting EZH1, I was able to determine that the band Read More …
For those following the USP5 project on my open notebook, you will recognize that assay development has been quite a challenge for screening ligands against USP5 zinc finger ubiquitin binding domain (Zf-UBD). In the past, I was unsuccessful in developing a differential scanning fluorimetry, and fluorescence polarization screening assay. I then tried 19F NMR spectroscopy screening, Read More …
The experimental set-up and results covered in this post can be found here: doi.org/10.5281/zenodo.1420537 I was previously using an HTT overexpression model to look at any changes in proteins related to HTT or its function. Unfortunately, none of the related proteins I looked at showed any significant change in protein levels with HTT overexpression. There Read More …
I used a medium-throughput 19F NMR assay to screen my second batch of compounds against USP5 zinc finger ubiquitin binding domain (Zf-UBD). To recap, the 19F NMR assay detects ligand binding by measuring perturbations in the resonance of a fluorinated tryptophan at the binding pocket. I selected the compounds for my second screen from SGC’s in Read More …
Searching far and wide for proteins that bind huntingtin
There are two major NSD3 isoforms expressed, long (aa 1-1437) and short (aa 1-645, differing in sequence from 620-645). The short isoform lacks a methyltransferase domain and, perhaps surprisingly, is the form attributed to NSD3’s role in acute myeloid leukemia (AML) [1]. I’ve previously used the TCGA-LUSC data-set [2] to test for differential expression based on the Read More …
Expression and purification of the HTT-HAP40 complex with expanded polyQ (Q54)
Following up on my previous post, I tested two antibodies I was planning on trying from Abcam and Novus that were used in previous publications. This time I tested directly in patient sample lysates and either saw no band, or many nonspecific bands (Figure 1). I have since reached out to the Orlando lab who Read More …
These days CRISPR gene editing needs little introduction. From humble origins as a bacterial defence system evolved to thwart viral invaders, CRISPR-based technologies have recently made headlines for their promise to revolutionize medicine. While there is a lot of excitement surrounding the use of gene editing in the clinic, it is also an incredibly valuable tool for studying the Read More …
