A detection antibody that specifically detects only MYPT1 phosphorylated at Thr696 and an HRP-conjugated secondary detection antibody is then applied.
Anti-mouse-HRP (Cell signalling 7076 S) was used as a secondary detection antibody.
However, cross-reaction of IgG with the secondary detection system can be excluded because the TrueBlot® system used here is designed to avoid reaction with denatured IgG, and control blots omitting the primary antibody showed no unspecific staining.
The proposed image-processing algorithm consists of a primary detection process based on binarization and a morphology analysis and a secondary detection process based on a convex hull.
m, Saturation of monoclonal antibody binding (in g i and j l experiments) was controlled by staining of blood-derived Tβ-syn cells with anti-VLA-4 or anti-LFA-1 primary monoclonal antibodies followed by secondary detection antibody (red) or with secondary antibody only (black).
Mouse monoclonal anti-human albumin antibody (ab399, Abcam) was used for primary detection (1 1000 at 37 °C for 2 h) and a horseradish peroxidase (HRP) goat anti-mouse IgG secondary antibody (405306, BioLegend, London, UK) was used for secondary detection (1 2000 at 37 °C for an hour).
Isolation and identification of Pathogens: Membrane filtration method is also used for the secondary detection of E. coli.
This will be followed by a secondary detection stage that uses slightly more information to recover the transmitted data.
Secondary detection was performed using anti-mouse (1 4000) or anti-rabbit (1 5000) (Bio-Rad, Hercules, CA) HRP conjugated antibodies.
A new methodology based on the co-localization of the primary captured antibodies and the secondary detection antibodies is now proposed, namely ACM or Antibody Colocalization Microarray.
Secondary detection was performed with the TSA™ kit #26 with streptavidin-HRP (Invitrogen, Carlsbad, CA, USA).
