Monoclonal hrp conjugated anti 6 his antibodies

The E. coli cells were disrupted by sonication, the cell lysates were centrifuged, and the supernatants were used for western blot. Protein concentration was determined using BCA protein assay kit (Pierce), and the same amount of protein sample was separated in 12% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and incubated with monoclonal HRP-conjugated anti-6×His antibodies (1:10,000 diluted) (Abcam). Protein signals were detected using Immobilon Western HRP substrate (Millipore) and X-ray film. To separate the phosphorylated proteins, 20–50 μM Phos-tag Acrylamide (WAKO) and 0.1 mM Mn²⁺ were added into the SDS-PAGE. Quantification was conducted using ImageJ software (NIH).

Immunoblot analysis was performed as described previously with small modifications [31 (link)]. Briefly, bacterial samples were disrupted by sonication (TissueLyser, China). The cell lysates were subsequently centrifuged, and the precipitates were transferred to membrane protein dissolution buffer (Sangon Biotech). The dissolved samples were used in western blotting. Protein concentration was determined using a BCA protein assay kit (Abcam, China). The proteins were transferred to nitrocellulose membranes (Bio-Rad, USA), and the membranes were blocked with 3% (w/v) milk (BD, USA) solution before incubation with monoclonal HRP-conjugated anti-6× His antibodies (Abcam, UK) diluted in 5% (w/v) BSA (Sangon Biotech). Protein signals were detected by HRP Substrate (Bio-Rad, USA) and Enhanced ECL Chemiluminescent Substrate (MKBio, China).