Alkaline phosphatase conjugated goat anti rabbit secondary antibody

Streptozotocin (STZ), gallic acid, andrographolide, neutral buffered formalin, Harris hematoxylin solution, eosin Y, and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody were purchased from Sigma-Aldrich (Selangor, Malaysia). Sodium azide and all organic solvents were purchased from Merck (Selangor, Malaysia). Rat Insulin ELISA kit was purchased from Mercodia (Sweden). CHOL2 total cholesterol test kit and TRIGL (GPO-PAP) triglyceride test kit were procured from Roche (Germany). RIPA lysis buffer, 2 × protein loading buffer, Tris-buffered saline with 0.1% (v/v) Tween-20, and BCIP/NBT chromogenic substrate kit were purchased from Solarbio (China). PVDF membrane was procured from Bio-Rad (USA). Rabbit anti-rat GLUT4 primary antibody was procured from Cusabio (China).

To explore the potential interaction between LvFLp53, LvΔNp53 and LvDorsal, pAc5.1-LvFLp53-FLAG or pAc5.1-LvΔNp53-FLAG was transfected with pAc5.1-LvDorsal-GFP or pAc5.1-GFP (as a control) into S2 cells. After 48 h, cells were harvested and lysed in Pierce IP lysis buffer (Thermo, USA) with proteinase inhibitor cocktail (Sigma, USA). The co-immunoprecipitations were performed using anti-FLAG tag agarose conjugated gel (Abmart, China) and anti-GFP tag agarose affinity gel (MBL International Corporation, Japan), respectively. Western blotting was performed with rabbit anti-GFP antibody (Sigma, USA) and rabbit anti-FLAG antibody (Sigma, USA), and alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (Sigma, USA). A standardized aliquot (5%) of each total input cell lysates was also examined as control.

Immunoblotting of the purified recombinant proteins was carried out as described earlier [44 ]. The proteins were separated by 12% SDS-PAGE and transferred to Hybond C membrane (Amersham Pharmacia Biotech, England). The transfer buffer consisted of 150 mM glycine, 20 mM Tris-HCl (pH 8.0), 0.1% SDS, 10% methanol. After the transfer, the membrane was stained with Ponceau S (Sigma-Aldrich, USA). After destaining in autoclaved double distilled water, the membrane was incubated for 2 h at RT in blocking buffer (200 mM Tris-HCl-pH 7.5, 1.4 M NaCl, 0.02% Tween-20, 3% BSA). The blots were washed thrice with TBST (200 mM Tris-HCl-pH 7.5, 1.4 M NaCl, 0.02% Tween-20), followed by TBS (200 mM Tris-HCl-pH 7.5, 1.4 M NaCl) for 10 min with each of the buffers. The blots were incubated with rabbit anti-His antibodies (Biovision, India) at 1:10,000 dilution in blocking buffer for 2 h for AtCyp19-3 proteins and for overnight for PpiA. The blots were washed thrice with TBST containing 0.2% Tween-20, followed by TBS for 10 min each. After washing, the blots were incubated in alkaline-phosphatase conjugated goat anti-rabbit secondary antibodies (Sigma-Aldrich, USA) (diluted 1:10,000 in TBS buffer) for 2 h. After washing the blot thrice with TBST and TBS for 10 min each, the protein-antibody complex was visualized by incubating in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution.