Finally, chromatin structure in HeLa cells was also analyzed in the SEVENS assay

ImageJ 2x″. Thioflavin T binding assay The INK-128 inhibition of A fibril formation by scFv was monitored via thioflavin T fluorescence. PubMed ID: In an A42 fiber formation inhibition assay, 50 M A42 was incubated with 250 M A8 scFv, and the samples were removed for detection at different time points. In the A42 fiber disaggregation assay, mature A42 fibers were incubated with a 10-fold molar excess of scFv in borate buffer at 37C for 24 and 48 h. The assay was performed with 20 M ThT, 0.1 M boric acid, 2.5 mM NaCl, and 2.5 mM sodium borate. To block the binding of scFv, the anti-A scFv was pre-incubated with A111 and A1224 peptides in the A aggregation and disaggregation assays, respectively. The fluorescence of the parallel reactions was measured with a Fluorescence Spectrophotometer F-7000 at 442 nm and 484.8 nm with a scan speed of 240 nm/min. The assay was performed three times. 6 / 16 Inhibiton of A Fibril Aggregation and Promotion of Disaggregation Statistical analyses The fibril length and ThT fluorescence data were analyzed using one-way or two-way ANOVAs and the chi-square test. An level of 0.05 was used for all statistical significance tests. Results Expression, purification and identification of anti-A scFv through rBacmid in baculovirus To express the variable region of MAb PubMed ID: A8 without the Fc fragment, the VL and VH genes were combined using the 3 linker sequence to form the VL-3-VH or VH-3-VL structures through a two-step SOE-PCR method. Our data showed that the linker sequence was introduced by the primer after the first PCR step. The 300 bp of the VL3, 3-VH, VH-3, and 3VL genes were amplified, which provided templates and primers for the following PCR. The 750 bp of the VL-3-VH and VH-3-VL genes were produced as a result of the second PCR step. To treat the A aggregation system with the scFv expressed in eukaryotic cells, we attempted to construct a rBacmid encoding anti-A scFv in the baculovirus expression system in which the viruses were packaged in Sf9 cells. According to our previous results from the E. coli system, the expression level of VL-3-VH was substantially higher than that of VH-3-VL; thus, the VL-3-VH orientation was selected for subsequent expression in Sf9 cells. The scFv gene with the His tag at the N terminus or at the C terminus was produced via PCR and then cloned into a baculovirus package vector. The recombinant plasmid pFastBac1-His-VL-3-VH or pFastBac1-VL-3-VH-His was identified via BamHI/ XhoI digestion. After transposition, the 3,000-bp M13 PCR products observed by agarose gel electrophoresis indicated that rBacmid-His-VL-3-VH and rBacmid-VL3-VH-His were correctly constructed. To identify the His-tagged scFv that was expressed in baculovirus, the proteins were first purified using a Ni-NTA affinity purification gel. The Coomassie brilliant blue staining showed that purified His-scFv and scFv-His were expressed at 30 kDa based on SDS-PAGE, which could be verified via Western blot analysis with an HRP-labeled anti-His antibody. To confirm the expression of anti-A scFv, the purified scFvs were analyzed using mass spectrometry. The 750 bp of the VL-3-VH and VH-3-VL genes were also cloned into the pET-30a vector and expressed in E. coli BL21 cells. Immunoreactivity of anti-A scFv expressed in baculovirus To identify the bioactivity of the scFv expressed in baculovirus, the antigen was detected using dot blot and indirect ELISA analyses. The dot blot results showed that all of the scFvs expressed in baculovirus