Supplementary Figure 33 DLS and SDS PAGE confirm AtzBSH2 incorporation into the 3-component assembly

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design Preprint: doi: 10.1101/274183 Fig S33. <b>DLS and SDS PAGE confirm AtzBSH2 incorporation into the 3-component 4 assembly.</b> AtzAM1, AtzBSH2, and AtzCM1 were added and allowed to incubate at various concentrations, then analyzed with DLS which showed that the addition of AtzBSH2 continues to have an assembly at ~1 µm. The SDS PAGE gel samples were pelleted and samples of the three component assembly supernatant and pellet were analyzed. If AtzBSH2 is incorporated into the assembly, it should partition preferentially into the pellet. The gels show that a band at the expected MW weight of AtzBSH2 ~69kda is seen predominantly in the pellet with increasing AtzBSH2 concentrations (Also see Fig S34). Standards for SDS-PAGE used are the BioRAD Precision Plus Protein Unstained Protein Standards. GelDoc EZ System Strain-free Imaging (BioRAD) was used to image and analyze the SDS-PAGE gel. Full gel with annotated ladder is provided for verification. (SI 2.9) Phosphorylation, assembly formation, and disassembly – The phosphorylation 14 protocol was based upon Src kinase activity assay by Sigma (Catalog # S1076). In a final 15 reaction volume of 150μL, 3μM AtzAM1 was mixed into 1X Kinase Activity Buffer (4mM MgCl2, 16 2.5mM MnCl2, 0.25mM DTT, 5mM MOPS, 2.5mM glycerol-2-phosphate, 1mM EGTA, 400nM 17 EDTA, pH 7.6), 2.5 mM MnCl2,HNG, 2 mM ATP, 800ng Src kinase, and incubated for 7 – 16 hr at 25°C for phosphorylation to occur. After phosphorylating, AtzCM1 was added to a final 2μM concentration. Assembly was allowed to form at 2hr 25°C. Disassembly was performed by adding 4.8μg of YopH phosphatase into the 150μL reaction mixture after assembly formation occurred. Size measurements using DLS were performed to determine assembly formation/disassembly (SI 2.10) Dynamic light scattering (DLS) – 50 μL of an assembly sample was used for size 25 determination using a Malvern Zetasizer and a quartz cuvette (ZEN2112, Malvern). Ten spectra measures were recorded for eleven replicates at 25 °C. The standard operating procedure accounted for 5% glycerol in solution.<br>