Borrelia burgdorferi Protein Co-IP

Co-IP experiments were performed using the Pierce Direct IP Kit (Thermo Scientific, Rockford, IL). Briefly, B. burgdorferi B31 cells were grown to mid-log phase (5 × 10⁷ organisms ml⁻¹) in complete BSK-II medium before whole-cell lysates were prepared as previously described (Lenhart et al., 2012 (link)). 500 µl of final lysates were then mixed with AminoLink resin with conjugated BB0794, BamA, or GST antibodies following the manufacturer’s instructions (Thermo Scientific). The lysate and resin mixture was rotated end over end at 4°C overnight before being washed. Proteins bound to the resin were eluted by adding 50 µl of final sample buffer containing β-mercaptoethanol and boiled for seven minutes before SDS-PAGE and immunoblot analysis.

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Iqbal H., Kenedy M.R., Lybecker M, & Akins D.R. (2016). The TamB ortholog of Borrelia burgdorferi interacts with the β-barrel assembly machine (BAM) complex protein BamA. Molecular microbiology, 102(5), 757-774.

Publication 2016

Pierce Direct IP Kit AminoLink resin

Antibodies B cells Buffer Cell Immunoblot analysis Mercaptoethanol Proteins Resin Sds page

Corresponding Organization : University of Colorado Colorado Springs

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Variable analysis

independent variables

Antibodies used for co-immunoprecipitation (BB0794, BamA, or GST)

dependent variables

Proteins bound to the resin and eluted

control variables

Cell growth to mid-log phase (5 × 10^7 organisms ml^-1) in complete BSK-II medium
Whole-cell lysates preparation as previously described
Rotation of lysate and resin mixture end over end at 4°C overnight
Washing of the resin after incubation
Elution of bound proteins by adding final sample buffer containing β-mercaptoethanol and boiling for seven minutes

controls

No positive or negative controls were explicitly mentioned in the provided information.

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