SDS-PAGE

Clean the components of the electrophoresis camera with 70% ethanol and gauze 's.

Assemble the chamber and check that there are no leaks by pouring distilled water between the glasses.

Prepare polyacrylamide gels:

A	B
Reagents	2 minigels
Distilled water	3.4 mL
Acrylamide/Bis 30%	4 mL
Tris-HCl/SDS 4X pH 8.8 (1.5 M Tris-HCl, 0.4% SDS)	2.5 mL
ASP 10%	100 µL
TEMED	4 µL

Separation gel (12%)

Pour the solution between the glasses with a 1 mL micropipette, leaving a space of 1.5 cm for the concentrating gel. To level, distilled water is added and allowed to settle for 0h 30m 0s or until a line is seen between the gel and the water.

A	B
Reagents	2 minigels
Distilled water	2.7 mL
Acrylamide/ Bis 30%	1 mL
Tris-HCl/ SDS 4X pH 8.8 (1.5 M Tris-HCl, 0.4% SDS)	1.3 mL
ASP 10%	50 µL
TEMED	4 µL

Concentrating gel (6%)

Pour solution onto separating gel using a 1 mL micropipette.

0h 30m 0s 0h 30m 0s Insert the comb (carefully avoiding the formation of bubbles) and leave to solidify for 0h 30m 0s

When the gels are polymerized, prepare an electrophoresis chamber with 1X running buffer until it covers the gels and it reaches the line of two gels.

Sample PTake the pellets contained in Eppendorf tubes

• Take the pellets contained in Eppendorf tubes
• Add 300µL of 1X loading buffer and resuspend the pellet.reparation:

To denature proteins, heat samples in boiling water for 5 min.

Load gels with the hot sample.

In the first well add 7µLof the molecular weight marker.

Once the samples are loaded, run the gel at 80 V for 0h 20m 0sand then at 180 V for 0h 45m 0s

Turn off the camera and disarm it.

Remove the gels from the glasses and place in a container with staining solution. Leave stirring for one hour.

Remove staining solution after one hour, add destaining solution and leave stirring for 0h 20m 0s Change the destaining solution and leave again for 0h 20m 0s

Leave stirring until the gel is transparent.

Analyze the gels.