MRSA was grown in ZnO-NRs at
different concentrations (1/8 percent MIC, 1/4 MIC, and 1/2 MIC) for
30 min, while the negative control was grown without ZnO-NRs. The
collected bacterial proteins were extracted and suspended in an extraction
kit (iNtRON iotechnology), including Tris-HCI (pH 7.5). According
to the manufacturer’s procedure, we used a Bio-Rad protein
assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) to elute the
protein concentrations. As a result, the supernatant was collected,
and aliquots of protein in equal amounts were placed in new tubes.
SDS-PAGE was used to separate proteins, which were then transferred
onto nitrocellulose membranes (Millipore, MA, USA) for 3 h at 250
mA at 4 °C using the Bio-Rad electroblotting system (Bio-Rad
Mini Trans-Blot Electrophoretic Transfer Cell). We used 5% skim milk
in Tris-buffered saline with Tween-20 buffer to block the membrane.
After blocking, the membranes were probed with monoclonal mouse anti-PBP2a
primary antibody and GPDH (diluted 1:1000, Bio-Rad, USA) and re-probed
with an anti-mouse IgG secondary antibody (diluted 1:2000, Enzo Life
Sciences, Ann Arbor, MI, USA). Then, the membranes were treated with
ECL PrimeWestern Blotting Detection Reagent (Invitrogen, USA), and
the bands were visualized with an Image Quant LAS-4000 mini chemical
luminescent imager (GE Healthcare Life Sciences).23 (link)
different concentrations (1/8 percent MIC, 1/4 MIC, and 1/2 MIC) for
30 min, while the negative control was grown without ZnO-NRs. The
collected bacterial proteins were extracted and suspended in an extraction
kit (iNtRON iotechnology), including Tris-HCI (pH 7.5). According
to the manufacturer’s procedure, we used a Bio-Rad protein
assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) to elute the
protein concentrations. As a result, the supernatant was collected,
and aliquots of protein in equal amounts were placed in new tubes.
SDS-PAGE was used to separate proteins, which were then transferred
onto nitrocellulose membranes (Millipore, MA, USA) for 3 h at 250
mA at 4 °C using the Bio-Rad electroblotting system (Bio-Rad
Mini Trans-Blot Electrophoretic Transfer Cell). We used 5% skim milk
in Tris-buffered saline with Tween-20 buffer to block the membrane.
After blocking, the membranes were probed with monoclonal mouse anti-PBP2a
primary antibody and GPDH (diluted 1:1000, Bio-Rad, USA) and re-probed
with an anti-mouse IgG secondary antibody (diluted 1:2000, Enzo Life
Sciences, Ann Arbor, MI, USA). Then, the membranes were treated with
ECL PrimeWestern Blotting Detection Reagent (Invitrogen, USA), and
the bands were visualized with an Image Quant LAS-4000 mini chemical
luminescent imager (GE Healthcare Life Sciences).23 (link)
