IGEM:IMPERIAL/EMSA

How to mesure LuxR

To the other biologists, remember disco dancing mice?

To everyone else

LuxR is a DNA binding Protein therefore we can purify the DNA bound to LuxR using an Electrophoretic mobility shift assay (EMSA) This tequnique relies on the fact that DNA bound by protein will migrate through a gel slower than DNA not bound to protein. Thereby we can cut the slower band of DNA from the first gel and run a second gel and purify LuxR and mesure it's concentration.

Rough Protocal

• Grow bacteria on radioactive media to get radioactive proteins
• lyse bacteria
• add crude lysate to a tube
• add a vast excess of AHL to the tube
• add a large excess of pure plasmid which contains LuxPR (for the DNA to bind)
• add non-specific competitor such as poly(dI∙dC) or poly(dA∙dT). to mop up non- specific binding to non-specific sites, this must be in large excess, (200fold)
• run on gel

• the slowest band should be LuxR/AHL + plasmid cut this out and run on a mass spec or 2D gel to get luxR concentratition

John.C

[Full Protocall] [Info on EMSA] [EMSA KIT]

Tom Says:
Easy to visualise the result
• Information on stoichiometry and cooperativity
• Not fast (~2 hrs prep, 2 hrs running time)
• Not an equilibrium method (dilution of sample occurs during run)
• Does not work well for weak interactions
• Only semi-quantitative
• Non-equilibrium conditions - cannot measure KD
• But can compare different proteins (mutants?) easily