Ecltm western blotting kit

Cells from 1 ml culture were centrifuged and 1X LDS sample buffer with reducing agent (Thermo Fisher) was added to the cell pellet, sufficient to give an OD₆₀₀ of 2.5. Samples were boiled and resolved on a NuPAGE 10% Bis-Tris Gel in MOPS SDS Running Buffer from Thermo Fisher. Proteins were transferred onto a nitrocellulose membrane with an iBlot gel transfer system. Lysine-acetylated proteins were detected with mouse anti-AcK antibody (Cell Signaling Technology, cat. # 9681S) diluted 1/1000 in TBS-T with 5% of BSA, incubated overnight at 4°C and developed with peroxidase conjugated anti-mouse antibody. The signal was detected with a chemiluminescent reaction, using ECL^TM Western Blotting kit from GE Healthcare, and detected by a BioRad Molecular Imager ChemiDoc XRS System.

The extracted soybean proteins were subjected to SDS–PAGE. Proteins in the 12.5% gel were stained with Coomassie brilliant blue (CBB) (CBB R-350, GE Healthcare) to visualize the total protein patterns. The immunoblotting analysis was conducted by transferring the SDS–PAGE gel to an Immobilon-P^TM PVDF membrane (Millipore) using a semi-dry blotting method [32 (link)]. The membrane was incubated in 10 mM PBS (pH = 7.5) containing 0.1% Tween-20 (PBST) and 5% skim milk for blocking. The membrane was then incubated for 1 h at room temperature (25 °C) in a blocking buffer containing allergen-specific antibodies. After the membranes were washed four times with PBST for 10 min, the bound primary antibodies were detected by using HRP-conjugated goat anti-rabbit, anti-mouse, or anti-guinea pig IgG and an ECL^TM Western blotting kit (GE Healthcare). The resultant chemiluminescent signals were detected on X-ray film (Hyperfilm^TM MP, GE Healthcare). Immunoblotting experiments were performed three times, and band densities were determined using Alpha Ease^TM software (Alpha Innotech, San Leandro, CA, USA). The immunoblot results were expressed relative to the value of the control No. 1 sample (C1).

OVA was subjected to SDS-PAGE [34 (link)]. Proteins in the 12.5% gel were stained with Coomassie brilliant blue (CBB) (CBB R-350, GE Healthcare) to visualize the total protein patterns. Immunoblotting analysis was conducted by transferring the SDS-PAGE gel to an Immobilon-P^TM PVDF membrane (Millipore) using a semi-dry blotting method [35 (link)]. The membrane was immersed in 10 mM PBS (pH 7.5) containing 0.1% Tween-20 (PBST) and 5% skim milk for blocking. The membrane was incubated overnight with mouse sera (1:100 dilution) at 4 °C. After washing with PBST three times for 10 min, the protein surface of the membrane was reacted with 1:50,000 dilutions of HRP-conjugated anti-mouse IgG1 antibody for 1 h. After washing the membranes four times with PBST for 10 min, the bound primary antibodies were detected using HRP-conjugated goat anti-mouse IgG1 and an ECL^TM Western blotting kit (GE Healthcare). The resultant chemiluminescent signals were detected on an X-ray film (Hyperfilm^TM MP, GE Healthcare).