Goat anti mouse igg hrp

Protein lysate preparation and western blotting were performed as previously described with the following change [62 (link)]. PVDF membranes were used and membranes were pre-wet in methanol for two minutes and then incubated in transfer buffer for five minutes. The following primary antibodies were used: rat anti-HSF1 mAb (Enzo Lifesciences, Farmingdale, NY), rabbit anti-HSP27 pAb (Enzo), rabbit anti-DNAJB1/HSP40β pAb (Enzo), rabbit anti-DNAJC17/HSP40C pAb (Abcam, Cambridge, United Kingdom), mouse anti-HSP70/72 mAb (Enzo), rat anti-HSP90α mAb (Enzo), mouse anti-HSP90β mAb, rabbit anti-HSP105/110 pAb (Enzo), rabbit anti-HSF1 phospho-serine (pS) 326 (Abcam), and rabbit anti-HSF1 pS303 (Abcam). The following secondary antibodies were used: ECL Rabbit IgG HRP-linked whole Ab (from donkey) (GE Healthcare), ECL Mouse IgG HRP-linked fragment Ab (from sheep) (GE Healthcare) [for all mouse antibodies except anti-HSP90β], goat anti-mouse IgG HRP (PerkinElmer Life Sciences, Boston, MA) [for anti-HSP90β], and goat anti-rat IgG HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Streptavidin 96-well microtiter plates (Invitrogen) were coated with 0.5 μg/ml biotinylated peptides in PBS. After overnight incubation at 4°C the plates were washed 3 times with PBS-T. Mouse serum samples were diluted 1:1000 with 1% BSA/PBS-T and 100 µl were added to the respective wells and plates were incubated 2h at 4°C. Serial dilutions of serum samples were also made to measure antibody titer against selected peptides. Wells were washed 5 times with PBS-T and 100 µl of 1:2000 diluted goat anti-mouse IgG/HRP (PerkinElmer) were added to each well. After 1 h incubation at 4°C plate wells were washed 5 times with PBS-T and 100µl of OPD were added to each well. Color development was observed, the reaction was stopped with 100 µl H₂SO₄ and plates were read at 492 nm using the HIDEX Sense plate reader. The ELISA’s cut-off level was set to twice the mean absorbance obtained from the blank wells.