Analysis of the degradation of amyloid beta fibrils after separation via the combination of non‐denaturing agarose electrophoresis and Congo red dye staining

Abstract

A combinational method for the separation and the degradation of amyloid‐β fibrils in the biological samples is developed to suppress the amyloid‐β fibrils content. The complexes of the Congo red dye and amyloid‐β fibrils migrate to the anode in a non‐denaturing agarose electrophoresis owing to its negative charge, thus separating the complex containing amyloid‐β fibrils from the human plasma proteins and the amyloid‐β monomer. The separated amyloid‐β fibrils exhibited birefringence under a polarizing microscope and immunoreactivity to the anti‐amyloid‐β antibody. The digestion of 0.23 nmol of the amyloid‐β fibrils using nattokinase at 37°C for 24 h increased with the amount of enzyme up to 9 pmol, beyond which the digestion was independent of the enzyme concentration. The digestion of 0.23 nmol of the amyloid‐β fibrils with 22.5 pmol of nattokinase at 37°C increased with time up to 6 h, following which, it became independent of time. The extraction of the separated amyloid‐β fibrils from the agarose gel accompanied the removal of major plasma proteins from the spiked plasma and amyloid‐β fibrils. The combination of the non‐denaturing agarose gel electrophoresis and the dye staining would be applicable to examine formation and degradation processes of amyloid‐β fibrils after their separation from the biological samples.

Characterization of enzymatic activity of lysozyme in lysozyme–ovotransferrin complex before and after treatment with trypsin

“…To identify the lysozyme and ovotransferrin, immunoblotting was performed as a previously reported [21,22]. Proteins separated by non‐denaturing 2‐DE and sodium dodecyl sulfate‐polyacrylamide gel (SDS‐PAGE) were transferred from the gel to a PVDF membrane using a semi‐dry transblotting apparatus for immobilization [23].…”

Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining

“…To identify the protein components of the protein complexes, immunoblotting was performed, as previously reported 11) . Proteins separated by non-denaturing 2DE or SDS-PAGE were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using a semidry transblotting apparatus for immobilization 12) . After blotting, the PVDF membrane was soaked in 5 mL of 1% w/v bovine serum albumin (BSA) in PBS for 1 h. A buffer containing 1% w/v BSA, polyclonal anti-carboxylesterase antibody (1/1000 dilution), or 1% w/v BSA and polyclonal transferrin antibody (1/1000 dilution) was added to the membrane, which was then incubated for over 2 h at 25°C.…”