P nitrophenyl phosphate n1891

Hb-α-syn complex levels in brain tissue, cell lysates, and RBCs were measured by ELISA using a mouse monoclonal anti-Hb antibody (ab77125; Abcam, Cambridge, MA, USA) and biotinylated mouse monoclonal anti-α-syn (3D5) antibody [40 (link)] for capture and detection, respectively. After completion of the immunoreaction, samples were incubated with 100 μl ExtrAvidin alkaline phosphatase (E-2636; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in blocking buffer followed by the enzyme substrate p-nitrophenyl phosphate (N1891; Sigma-Aldrich). The reaction was allowed to proceed for 30 min at room temperature, after which the absorbance was read at 405 nm using a Multiskan MK3 microplate reader (Thermo Scientific, UT, USA).

The oligomeric α-syn concentration in brain tissues and cell lysates was measured by ELISA [18 (link), 42 (link)] using non-biotinylated and biotinylated 3D5 mouse monoclonal antibodies [40 (link)] for capture and detection, respectively. After completion of the immunoreaction, the contents of each well of the ELISA plate were incubated with 100 μl of ExtrAvidin Alkaline Phosphatase (E-2636; Sigma-Aldrich, St. Louis, MO, USA) diluted at a ratio of 1: 20,000 in blocking buffer, and then reacted with the enzyme substrate p-nitrophenyl phosphate (N1891; Sigma-Aldrich, St. Louis, MO, USA). The reaction was allowed to proceed for 30 min at room temperature, after which the absorbance was read at 405 nm using a Multiskan MK3 microplate reader (Thermo Scientific, UT, USA).
To detect pS129 α-syn, 0.1 μg/ml of anti-pS129 α-syn polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in coating buffer was used for capture. The remaining steps were the same as for the detection of oligomeric α-syn by ELISA.