Mini protean tgx precase gel

Proteins were separated on a 12% Mini-Protean TGX Precase Gel (BioRad) before being incubated overnight in GelCode (Thermo Fisher). Gels were washed with deionized water and imaged using GeneSys.

Proteins were separated on a 12% Mini-Protean TGX Precase Gel (BioRad) before being transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 10% (wt/vol) non-fat milk buffer in 1× DPBS and then incubated with anti-IpaB, anti-IpaC, and anti-IpaD antibody (1:10,000) (kindly donated by the Wendy Picking Laboratory) diluted in 10% (wt/vol) non-fat milk buffer overnight at 4°C on an orbital shaker. Following washing, the membrane was incubated with secondary goat anti-rabbit 680 nm (Thermo Fisher), and proteins were visualized using 700-nm wavelength setting on a LI-COR Odyssey Laser Scanner. Whole cell lysates were normalized using DnaK (Invitrogen) as a loading control, and densitometry (ImageJ) (66 (link)) was utilized to compare loaded protein levels. There is no other defined protein in Shigella that is secreted in a consistent quantity for use as a control. Densitometry (ImageJ) for secreted fractions were determined by assuming 2457T without bile salts was 1 and comparing all secreted levels protein levels to it.