A8 35 amphipol

When BamA protein was required for ELISA or antigen-based sorting, an in vitro biotinylation reaction using BirA enzyme targeting the N-terminal Avi tag was first carried out according to the manufacturers suggestions (Avidity); BamA was then rerun over the Superdex 200 (26/60) in buffer E as described above in order to remove free biotin and other reaction components. When protein was required in amphipol (either non-biotinylated or biotinylated), BamA at 1 mg/mL in buffer E was incubated with a stock solution of 2 mg/mL A8-35 amphipol (Anatrace) at 22 °C for 1 hour and then applied over a Superdex 200 (26/60) column in buffer F (50 mM Tris pH 8, 100 mM NaCl). Protein reconstituted in A8-35 amphipol for immunization was concentrated to 1 mg/mL using a centrifugal device (10 K MWKO; Millipore). When PE-labeled protein was required for antigen-specific sorting, BamA at 1 mg/mL (biotinyated and reconstituted in A8-35 amphipol in buffer F) was mixed 1:1 (vol/vol) with PE-streptavidin (Jackson ImmunoResearch) reconstituted in buffer F; the PE-streptavidin-biotin-BamA-amphipol complex was incubated at 22 °C for at least 15 minutes prior to use.

When LptDE protein was required for ELISA or antigen-based sorting, an in vitro biotinylation reaction using BirA enzyme targeting the N-terminal Avi tag was first carried out according to the manufacturer's suggestions (Avidity); LptDE was then rerun over the Superdex 200 (26/60) in buffer E as described above in order to remove free biotin and other reaction components. When protein was required in amphipol (either non-biotinylated or biotinylated), LptDE at 1 mg/mL in buffer E was incubated with a stock solution of 2 mg/mL A8-35 amphipol (Anatrace) at 22°C for 1 hr and then applied over a Superdex 200 (26/60) column in buffer F (50 mM Tris pH 8, 100 mM NaCl). Protein reconstituted in A8-35 amphipol for immunization was concentrated to 1 mg/mL using a centrifugal device (10 K MWKO; Millipore). When PE-labeled protein was required for antigen-specific sorting, LptDE at 1 mg/mL (biotinyated and reconstituted in A8-35 amphipol in buffer F) was mixed 1:1 (vol/vol) with PE-streptavidin (Jackson ImmunoResearch) reconstituted in buffer F; the PE-streptavidin-biotin-LptDE-amphipol complex was incubated at 22°C for at least 15 min prior to use.