Hrp conjugated goat anti mouse igg h l antibody

Cells were plated in a 100-mm culture plate with 8 mL of media. For immunoblotting analysis, 20 μg of protein was resolved with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel and transferred to polyvinylidene difluoride membranes (GE Healthcare Life science, Pittsburgh, PA, USA). The membranes were probed with anti-CD9 antibody (EPR2949, rabbit monoclonal, ab92726, Abcam, Cambridge, UK), anti-CD63 antibody (MX-49.129.5, mouse monoclonal, ab193349, Abcam), and anti-beta-actin antibody (C4, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin (IgG) antibody, HRP-conjugated goat anti-mouse IgG F(ab')₂ (both from Enzo Life Sciences, Farmingdale, NY, USA), and HRP-conjugated goat anti-mouse IgG (H+L) antibody (#1706516, Bio-Rad Laboratories, Hercules, CA, USA). The images were detected using an ECL chemiluminescent substrate (GE Healthcare Life Science) and analyzed using a LAS-3000 imager (Fujifilm, Japan).

Parasite homogenates (10 μg protein) were resolved on a 4–20% Mini-PROTEAN^® TGX^TM sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA). Protein was then transferred to an activated semi-dry polyvinylidene difluoride (PVDF) membrane, and the membrane was incubated in blocking buffer (phosphate buffered saline (PBS) + 0.05% Tween-20 (PBST) + 5% dry non-fat milk powder) at room temperature for 1 h. The membrane was incubated with anti-SmPGM or control antisera (generated as described below) diluted 1:10,000 in blocking buffer overnight at room temperature. The membrane was then washed with PBST and incubated with HRP-conjugated goat anti-mouse IgG (H + L) antibody (#170-6516, Bio-Rad) diluted 1:5000 in blocking buffer at room temperature for 1 h. The blots were developed using ECL Western Blotting Detection Reagents (GE Healthcare Bio-Sciences, Piscataway, NJ, USA), following the manufacturer’s instructions. Western blot images were captured using a ChemiDoc Touch Imaging System (Bio-Rad). An equivalent western blot probed with serum from mice immunized with adjuvant alone served as a negative control.

HaCaT and A549 cells were treated with gZnNPs (0, 25, and 35 μg/ml) for 24 h, and after exposure, the cell lysate was prepared in RIPA buffer (ab156034). The cell lysate was centrifuged at 13000 rpm, 4°C for 30 min, and the supernatant was used for protein expressions. The concentration of protein was evaluated by the Bradford method [11 (link)]. Protein (20 μg) was migrated on the gel and transferred to a PVDF membrane (Bio-Rad, Laboratories Inc., Berkeley, CA, USA). The PVDF membrane was incubated with different mouse monoclonal antibody against β-actin (1 : 12000 dilutions, Abcam, Cambridge, UK), Bcl2 (1 : 500 dilutions, Santa Cruz), Bax (1 : 1000 dilutions, Antibodies-online), Caspase-3 (1 : 500 dilutions, Cayman), and TNF-α (1 : 500 dilutions, Santa Cruz) for 24 h at 4°C.
Secondary antibody HRP-conjugated goat anti-mouse IgG (H + L) antibody (1 : 2000 dilutions Bio-Rad) was used. Immunoreactive bands were detected using an EZ west Lumi plus (ATTO Corporation, Tokyo, Japan), which is a chemiluminescent substrate to detect HRP on the western blotting membrane. The luminescence intensity (optical density) of the target protein bands was quantified using Lumino Graph 2 (ATTO Corporation). All protein expression levels were normalized to the levels of β-actin protein expression in each band.