Cy5 labeled secondary antibody

Cells were grown to OD₆₀₀ ~0.5 and a 1.5-mL aliquot was withdrawn, washed twice with 1 mL of PBS (50 mM NaH₂PO₄, pH 7.4). At each washing step, cells were centrifuged at 6,000 × g for 5 min at room temperature. After incubating 1 mL of washed cells at 35°C for 10 min, disulfide formation was promoted by the addition of 5 μL of a 60 mM Cu²⁺-phenanthroline solution (60 mM CuSO₄, 200 mM 1,10-phenanthroline, 50 mM NaH₂PO₄, pH 7.4) to yield a final concentration of 300 μM Cu²⁺. Samples were kept at 35°C for 10 min, after which the reaction was stopped by adding EDTA to a final concentration of 10 mM. Cells were pelleted by centrifugation at 21,000 × g for 5 min, and then lysed by resuspension in 100 μL of 1X TruPAGE™ LDS Sample Buffer (Sigma-Aldrich). Protein lysates were separated in an 8-12% TruPAGE™ gradient gel (Sigma-Aldrich) using TruPAGE™ Tris-MOPS SDS Express Running Buffer (Sigma-Aldrich). CheW-oligomers were detected by western blot using a polyclonal anti-HA antibody (ThermoFisher, catalog no. PA1-985) and a Cy5-labeled secondary antibody (ThermoFisher, catalog no. A10523). CheA and Tsr proteins were detected in similar fashion with appropriate polyclonal rabbit antibodies. Immunoblots were imaged under fluorescence mode in a Typhoon FLA 9500 scanner (GE Healthcare) and bands were quantified with ImageQuant software (GE Healthcare).