Team:EPF-Lausanne/Protocols/SDS-PAGE

 

SDS-PAGE protocol

This protocol is designed to make an SDS-PAGE on proteins purified directly from an overnight cell culture. There is no need to make in vitro protein synthesis.

Protein purification Lysis Buffer (2x) : for 100 ml Make an overnight culture of the cell containing your protein of interest
 * 4g of SDS
 * 20ml of glycerol
 * 2ml of 2-Mercaptoethanol
 * 70ml of stacking buffer (0.5 M Tris-HCl, pH=6.8)
 * 100mg of Bromophenol blue
 * 8ml of dd water

1. Pellet the bacteria from the overnight culture @ 4000 rpm for 30 min. Do the centrifugation @ 4ºC 2. Discard the supernatant 3. Re-suspend the bacteria in 1 ml of dd water. Centrifiguate for 30 min/4000 rpm @ 4ºC 4. Discard the supernatant 5. Add approximately 1 equivalent of Lysis buffer to your cell pellet 6. Heat the solution for 10 min @ 95ºC 7. Add 1 equivalent of dd water 8. Heat for 10 min @ 95ºC 9. Centrifuge @ 11600 g for 10 min -->Take the supernatant and apply the desired volume to a polyacrylamide gel (10 %) Note : for coomassie blue staining, 5ul of the solution should be enough to have well-stained bands Note : for speeding the processes of staining and de-staining, you can put your gel+solution in the microwave for the couple of seconds. CAUTION to the MeOH vapors which are highly toxic. You should perform this process under a negative pressure fume hood.
 * Stain your gel wiht a coomassie blue solution (in MeOH and AcOH)-->incubate with soft shaking @ RT for about 2h
 * De-stain your gel using a 10% AcOH and 50% MeOH in water. The solution has to be changed every time it gets blue. You might need ot change the solution up to 20 times.