Anti rabbit ap conjugated igg

1´ SDS (sodium dodecyl sulphate) sample buffer (New England BioLabs) per 1 ml of OD600 of 1.0 cells in 200 µl sample buffer. 5-10 µl of boiled supernatant of whole cell lysates was resolved on 4-12% Bis-Tris NuPAGE protein gel (Invitrogen) in 1´MES buffer (Invitrogen) at 150 V for 1 h. Resolved protein samples were transferred onto a PVDF membrane using the iBlot 2 dry blotting system (ThermoFisherScientific). Incubation of the membrane with primary and secondary antibodies was done at room temperature for 1 h each. Before and after secondary antibodies, the blot was washed 3´10 min with PBS+0.1% Tween 20. Western signals were visualized using CDP-star substrate (Invitrogen) on a Bio-Rad ChemiDoc MP Imaging system.
Antibodies were used at the following dilutions: mouse monoclonal anti-RFP antibody (MA5-15257) (1:3000; ThermoFisherScientific), monoclonal anti-FLAG M2-alkaline phosphatase antibody (1:3000; Sigma, A9469 ), monoclonal mouse anti-EF-Tu antibody (1:10,000; LSBio), polyclonal rabbit anti-Hfq antibodies (1:2500) (29), polyclonal rabbit anti-His antibodies (sc-803) (1:1000, Santa Cruz Biotechnology), anti-rabbit AP-conjugated IgG (1:5000; Cell Signaling Technology), and anti-mouse AP-conjugated IgG (1:10,000; Santa Cruz Biotechnology).