Bacterial Pulldown with soluble receptors

Materials

PBS (1x) – 24g NaCl, 0.6g KCl, 3.45g Na2HPO4*7H2O, 0.6g KH2PO4, pH 7.4, ad 1l A.bidest
Cell culture supernatants (e.g. OptiMEM) harvested from 293cells transiently transfected with constructs encoding soluble CEACAM- ectodomains fused to GFP
Polyclonal rabbit anti-GFP antibody (AG Hauck)
2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v) β-Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) Glycerin

Procedure

In case of low-affinity interactions (HopQ, UspA1, OMP P1) cluster the CEACAM- GFP protein before the Pulldown:

For each sample 1ml of soluble receptor supernatant is incubated with 1µl polyclonal GFP antibody rotating, over night at 4°C.

In case of high-affinity interactions (Opa proteins): there is no need to cluster the CEACAM-GFP protein before Pulldown. Supernatants can be used straight with the bacteria.

Bacteria are grown overnight for 18 h on LB agar plates. Bacteria are lifted from plates with a sterile cotton-tip, suspended in PBS and their OD₆₀₀ (Ecoli, Hemophilus, Moraxella) or OD₅₅₀ (Neisseria) is meassured. Bacteria (OD₆₀₀ 0.2) are suspended in cell culture supernatant containing the indicated receptor protein. Bacteria are incubated with equal amounts of the soluble receptor domains for 1 h at 20 °C with head-over-head rotation. After incubation, bacteria are centrifuged at 7000rpm, 5min and subsequently washed twice with PBS and either boiled in SDS sample buffer prior to SDS-PAGE and Western blotting or taken up in PBS and analysed by flow cytometry.