Journal: bioRxiv

Article Title: High-affinity nanobodies as tools for structural and functional studies on mammalian Arc

doi: 10.1101/2021.12.16.472929

Figure Lengend Snippet: A Analytical SEC demonstrated Nb binding to FLrArc-7A. 2.2 nmol of FLrArc-7A were mixed with an equimolar amount of Nb, incubated on ice for around 30 minutes before the whole solution (100 μ L) was injected on an Superdex 200 Increase 10/300 GL SEC column in 20 mM Tris, 150 mM NaCl, pH 7.5. 2.2 nmol of the unbound protein (apo) and Nbs alone were run as controls. B NiNTA Nb pulldown assay with equimolar amounts of His-tagged hArc-CTD (CTD) or MBP-2rNT (NTD) and untagged Nbs, followed by SDS-PAGE analysis. All Nbs associated with the CTD (upper panel), as noted by the presence of the Nb band in all complex elutions, whereas none bound to the NTD (lower panel). Pre denotes the complex prior to loading on the matrix, UB the unbound fraction, W1-3 the three washing steps, and Elu the eluted fraction.

Article Snippet: The protein was concentrated to 2 mL in a 30 kDa MWCO spin concentrator and applied onto a HiLoad 16/600 Superdex 200 pg size exclusion chromatography (SEC) column (GE healthcare, IL, USA) equilibrated in 20 mM Tris-HCl pH 7.4, 150 mM NaCl.

Techniques: Binding Assay, Incubation, Injection, SDS Page

Journal: bioRxiv

Article Title: High-affinity nanobodies as tools for structural and functional studies on mammalian Arc

doi: 10.1101/2021.12.16.472929

Figure Lengend Snippet: a) Analytical SEC of capsid hArc-Nb complexes. 2 nmol of Arc capsids were mixed with a 1.5-fold molar excess of Nbs and run on a Superdex 200 Increase 10/300 GL in PBS buffer. Noted is the presumably dimeric peak present in some Nb complexes, with an elution volume corresponding to that of the FLrArc-7A complexes. NbArc-E5 was accidentally mixed with 4 nmol of hArc, resulting in the large void peak and lack of Nb excess peak. b) SDS-PAGE analysis of SEC fractions, demonstrating that Nbs elute with the capsids in the void peak. c) Negative staining TEM images of capsid hArc at 150,000x magnification in complex with NbArc-H11 (left) and without Nb (right). Arrows point at capsids, and a magnified views of selected capsids is shown.

Article Snippet: The protein was concentrated to 2 mL in a 30 kDa MWCO spin concentrator and applied onto a HiLoad 16/600 Superdex 200 pg size exclusion chromatography (SEC) column (GE healthcare, IL, USA) equilibrated in 20 mM Tris-HCl pH 7.4, 150 mM NaCl.

Techniques: SDS Page, Negative Staining