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. Author manuscript; available in PMC: 2018 Oct 16.
Published in final edited form as: Cold Spring Harb Protoc. 2018 Sep 4;2018(9):pdb.prot100958. doi: 10.1101/pdb.prot100958

FIGURE 1.

FIGURE 1.

Schematic diagram of the protocol. (A) Preparation of the flow cell. (B) Immunoprecipitation (IP) and immunodepletion (ID) using Protein A Dynabeads and Xenopus egg extracts, followed by the branching microtubule nucleation assay via TIRF microscopy. (C) IP samples obtained using IgG, anti-HAUS1 (αH1), -anti-HAUS2 (αH2), and anti-HAUS3 (αH3) antibodies are analyzed by western blot to quantify the intensities of augmin subunits (H1-8). (D) Quantitative LC MS/MS using IP samples. Sequential mass spectrometry analyses allow identifying augmin subunits and relative amounts in each IP sample. (E) Hierarchical clustering dendrogram is generated by hclust package of R using normalized intensities of augmin subunits in each sample. Seven augmin subunits are depicted as H1-7 (H8 is not identified), showing a putative assembly model for augmin.