Journal: Cells

Article Title: A Nanobody-Based Toolbox to Probe ApoE4 in the Secretory Pathway and Cytosol

doi: 10.3390/cells15050479

Figure Lengend Snippet: Domain-specific binding of apoE nanobodies to apoE4. ( A ) Schematic representation of apoE structure. The N-terminal domain (residues 1–167) and C-terminal domain (residues 206–299) are connected by a hinge region. The N-terminal domain contains the low-density lipoprotein (LDL) receptor-binding region, while the C-terminal domain contains the lipid-binding region. The three human apoE isoforms differ at residues 112 and 158. Figure adapted from ref. , with permission from Springer Nature, 2018. Two apoE4 fragments corresponding to the N-terminal domain ( 1 ; aa1–173) and to the C-terminal domain ( 2 ; aa170–299) were produced. ( B ) Co-IP assays showing interaction of the six selected HA-tagged nanobodies with either the N-terminal apoE4 fragment (top; Coomassie-stained SDS-PAGE gels) or the C-terminal apoE4 fragment (bottom; Western blots). Co-IPs were performed using anti-HA agarose beads. The C-terminal apoE4 fragment was produced as an MBP fusion protein to stabilize the small fragment (~14 kDa). Samples were run on multiple gels, indicated by a space between the blocks. On each gel, 3 µg of apoE4(aa1–173) or MBP-apoE4(aa170–299) was loaded as a reference, together with negative control (NC) samples consisting of the respective apoE4 fragment incubated with anti-HA agarose beads in the absence of nanobody. For the N-terminal apoE4 fragment, a positive control (PC) was included in which the fragment was incubated with an anti-apoE antibody and Amsphere beads. The band seen at the nanobody level in the PC lane (top panel, lane 2) is background. For simplicity, one reference and one NC sample are presented. Uncropped gels and Western blots are shown in . ( C ) Subdivision of the six apoE nanobodies according to the apoE4 fragment they bind. Nanobodies 9-74 and 19-38 bind the N-terminal apoE4 fragment (aa1–173), whereas nanobodies 9-8, 17-69, 18-27 and 18-91 bind the C-terminal apoE4 fragment (aa170–299).

Article Snippet: As a positive control, Amsphere beads were incubated with 2 μg anti-apoE antibody (Novus Biologicals, Centennial, CO, USA, cat. no. NB110-60531), and the co-IP was performed as described above.

Techniques: Binding Assay, Produced, Co-Immunoprecipitation Assay, Staining, SDS Page, Western Blot, Negative Control, Incubation, Positive Control

Journal: STAR Protocols

Article Title: Standardized immunoprecipitation protocol for efficient isolation of native apolipoprotein E particles utilizing HJ15.4 monoclonal antibody

doi: 10.1016/j.xpro.2023.102271

Figure Lengend Snippet:

Article Snippet: WUE-4 Mouse monoclonal anti-apoE antibody (1:1000) , Abnova , Cat#MAB2371; RRID: AB_10548887.

Techniques: Control, Recombinant, Protease Inhibitor, Purification, Coomassie Assay, Software, Sterility