1 step ultra tmb blotting chromogenic substrate

Proteins were separated by SDS-PAGE using NuPAGE 4–12% (w/v) Bis-Tris gradient gels (Invitrogen). Silver staining was performed using the SilverQuest Silver Staining Kit (Invitrogen) according to the manufacturer’s protocol. For Western detection, proteins were transferred onto a PVDF membrane using the iBlot 2 dry blotting system (Invitrogen). The membrane was blocked in 3% (w/v) bovine serum albumin (BSA) dissolved in 1x PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.05% (v/v) Tween 20). As primary antibody rabbit α-GFP (abcam, ab290) was used, followed by secondary antibody HRP-conjugated anti-Rabbit IgG (ICL) incubation. The membrane was developed using the 1-Step Ultra TMB-Blotting chromogenic substrate (Thermo Scientific).

Proteins were separated by SDS-PAGE using NuPAGE™ 4-12% (w/v) Bis-Tris gradient gels (ThermoFisher Scientific Inc., Waltham, MA, USA). For Western detection, proteins were transferred onto a PVDF membrane using the iBlot™ 2 dry blotting system (ThermoFisher Scientific Inc., Waltham, MA, USA). The membrane was blocked in 3% (w/v) bovine serum albumin (BSA) dissolved in 1x PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.05% (v/v) Tween 20). Western blots were reacted with anti-HapX antisera (1:10,000; Davids Biotechnologie, Regensburg, Germany) or with polyclonal anti-c-Myc (1:1000; ab9106 Abcam plc, Cambridge, UK) as primary antibodies and with monoclonal anti-rabbit IgG peroxidase (1:10,000; A1949 Merck KGaA, Darmstadt, Germany) as secondary antibody. The membrane was developed using the 1-Step™ Ultra TMB-Blotting chromogenic substrate (ThermoFisher Scientific Inc., Waltham, MA, USA).