Horseradish peroxidase conjugated anti rabbit immunoglobulin g antibody

Protein extracts (30 μg) used for 2-DE were separated by 12% or 15% SDS-PAGE and then transferred to a nitrocellulose membrane. The residual binding sites on the membrane were blocked by treatment with 5% dry milk in TBS-tween 20. After milk saturation the membrane was incubated overnight at 4°C with 1:200 diluted primary rabbit polyclonal antibody against ABRACL, 1:200 diluted primary rabbit polyclonal antibody against FGB, 1:1000 diluted primary rabbit polyclonal antibody against ANXA3, 1:300 diluted primary rabbit polyclonal antibody against PGAM2. The membrane was washed three times in TBST for 10min, and then incubated for 90min at 4°C with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Sigma-Aldrich; Merck KGaA) at 1:3.000 dilution. Protein expression was visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc.), and the intensity of the signals was quantified by VersaDoc Imaging System (Bio-Rad Laboratories, Inc.). The intensities of the immunostained bands were normalized with the protein intensities measured by Coomassie blue (Sigma-Aldrich; Merck KGaA) from the same blot.

Heterologously expressed purified sHsp protein was fractioned on SDS-PAGE (12%) gel, and proteins were transferred to polyvinylidene diflouride (PVDF) membrane. The membrane was incubated for 1 h in 3% blocking solution (3% w/v skim milk in PBST: 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO4 with 0.5% Tween 20) at room temperature (RT). The protein-bound membrane was further incubated for one hour with polyclonal antiserum raised in rabbit against sHsp (diluted 1:5000 in PBST) at RT. The membrane was washed with PBST three times at RT/5min and then incubated with horseradish-peroxidase-conjugated anti-rabbit immunoglobulin G antibody (1:10,000; Sigma-Aldrich, St. Louis, MO, USA) at RT/1 h. For signal development, the membrane was incubated with HRP substrate (Immobilon^® Forte) in the dark for 1 min/RT, and signals were captured using a chemiluminescent detector (Azure Biosystems C300, Dublin, CA, USA).

Phosphoprotein extracts (20 μg) from IMAC columns used for 2-DE were separated by 12% and then transferred to a nitrocellulose membrane. The Western blotting procedure for phosphoproteins was conducted as previously described [21 (link)]. The membrane was blocked by treatment with 5% BSA in TBS-tween 20. After BSA saturation the membrane was incubated overnight at 4 °C with 1:200 diluted primary rabbit polyclonal antibody against Endoplasmic reticulum chaperone BiP (HSPA5), with 1:300 diluted primary rabbit polyclonal antibody against heat shock protein beta-1 (HSPB1), and 1:800 diluted primary rabbit polyclonal antibody against Vinculin (VINC). The membrane was washed three times in TBST for 10 min, and then incubated for 90 min at 4 °C with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA, Burlington, VT, USA) at 1:3.000 dilution. Protein expression was visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the intensity of the signals was quantified by VersaDoc Imaging System (Bio-Rad Laboratories, Inc.). The intensities of the immunostained bands were normalized with the protein intensities measured by Red Ponceau (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA, Burlington, VT, USA) from the same blot.