Anti mouse igg antibody

Western blot analysis was performed as previously described [15] , using the purified anti-Prep antibody (52 ng/ml at a final concentration [15] ) and the anti-Actb polyclonal antibody (1:1000 dilution, GTX109639, Genetex, Irvine, CA) as primary antibodies. As secondary antibodies, goat anti-chicken IgY antibody (1:10,000 dilution, 103-035-155, Jackson ImmunoResearch, West Grove, PA) and anti-mouse IgG antibody (1:5,000 dilution, NA934V, GE healthcare, Piscataway, NJ) were used for Prep and Actb, respectively. The signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck, Darmstadt, Germany) and quantified by the Image J software [35] to normalize the Prep band intensity to Actb. To confirm the specificity, we also performed this experiment by using the anti-Prep antibody preincubated with an excess amount of recombinant Prep protein.

The total protein concentration contained in the cell lysates and the brain homogenates was measured using the BCA Protein Assay Kit (Pierce). To digest PrP C , the samples were reacted with 20 µg/ml Proteinase K (PK) for 30 min at 37 C. After denaturation by SDS sample buffer (50 mM Tris-HCl pH 6.8, 5% glycerol, 1.6% SDS, 100 mM dithiothreitol) for 10 min at 95 C, 100 µg (cell lysates) or 50 µg (brain homogenates) of each sample was applied to 15% acrylamide gel for SDS-PAGE. The protein bands were then transferred to PVDF membrane. For blocking, the membrane was placed in 5% (w/v) skim milk with TBST (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 0.1% Tween 20) for 1 hour. For PrP detection, the membrane was then incubated with primary antibodies: M-20 (Santa Cruz Biotechnology) or SAF83 (SPI-Bio) diluted with 1% skim milk. Then, the membrane was further reacted with secondary antibodies: horseradish peroxidase-conjugated anti-goat (Santa Cruz Biotechnology) or anti-mouse IgG antibodies (GE Healthcare Life Sciences) diluted with 1% skim milk, and the bands were visualized using the ECL Prime Western Blotting Detection Kit (GE Healthcare Life Sciences) or Clarity Wester ECL Substrate (BioRad). The intensity of each band was quantified using the ImageJ software (National Institutes of Health).