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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}}..."

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Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=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}}..."

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{{Protokoll-Mix|titel=Materials|Inhalt=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.

Bacterial Pulldown with soluble receptors - Revision history