Ap conjugated anti rabbit igg secondary antibody

Protein samples collected from day 2 of immunoprecipitation were subjected to electrophoresis in polyacrylamide gel and transferred to polyvinyl difluoride membrane using a semidry apparatus as described previously.³¹ (link) The membrane was then blocked with 5% nonfat dry milk for 1 hour at room temperature. To determine LOX binding with FN or Coll IV in total protein extracts or ECM-only protein, membranes were subsequently incubated overnight at 4°C with rabbit anti-Coll IV antibody (1:1000, Catalog No. ab6586; Abcam) and rabbit anti-FN antibody (1:1000, Catalog No. ab2413; Abcam) followed by alkaline phosphatase (AP)-conjugated anti-rabbit IgG secondary antibody (1:1000, catalog no. 7054; Cell Signaling). Similarly, to assess extracellular LOX expression, membranes were incubated overnight at 4°C with rabbit polyclonal LOX antibody (1:2000, Catalog No. NB110; Novus) followed by AP-conjugated anti-rabbit IgG secondary antibody (1:1000, catalog no. 7054; Cell Signaling). The amount of protein loaded in the gel lanes was confirmed by β-actin antibody (1:1000, Catalog No. 4967; Cell Signaling). Densitometric values were determined to ascertain differences in protein levels using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).