I'm using his tag to measure the type and amount of proteins that secreted outside E. coli cells through Auto-transporter (surface display system). How I can prepare my sample for gel electrophoresis to be contain (only surface proteins, only cytoplasmic proteins, both surface and cytoplasmic proteins) ?
I think the secreted Proteins you can Screen by Screening the growth media.
surface Proteins by Membrane preperation
cytosolic by Lysis!
E. coli cells are disrupted by any of various physical means such as sonication, French press, or bead beater in a buffer containing protease inhibitor cocktail. The lysate is centrifuged at a relatively low g-force, like 10,000 x g for 20 minutes to pellet unbroken cells. The supernatant is then ultracentrifuged at 100,000 x g for 1 hour. The pellet contains the combined inner and outer membranes. The supernatant contains the cytoplasm.
The inner membranes can be dissolved with 1% Sarkosyl detergent. Ultracentrifuging again gives a pellet consisting of the outer membranes and a supernatant containing the detergent extract of the inner membranes.
Secreted proteins can be quantified by an E. coli total protein ELISA from the supernantant after removing the cells. After concentration, the quality/species can either be detected by 2-D gel separation followed by total protein staining or 2-D Western Blot (more sensitive).
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