Protocol E.2

Electroelution of Proteins From SDS-PAGE Gels

This is a general protocol that was developed for the ISCO electroeluter but could easily be applied to other systems.

Solutions

Gel Destain Solution

0.25 M EDTA 8.0 50 ml 500 mM EDTA 8.0

0.25 M Tris 9.0 25 ml 1M Tris pH 9.0

25 ml Q

Electroelution Buffer

20 mM Tris base 2.42 g Tris base

150 mM glycine 11.25 g glycine

0.01% SDS 0.1 g SDS

up to 1 liter with Q

HEGK10

20 mM HEPES 7.9 20 ml 1M HEPES pH 7.9

0.1 mM EDTA 0.2 ml 500 mM EDTA

20% glycerol 200 ml glycerol

10 mM KCl 0.75 g KCl

up to 1 liter with Q

 

 

Procedure

• Run a standard SDS-PAGE gel and stain with CuCl2 (Protocol C.2) to visualize the protein band(s).

• Excise the band and destain in 3 x 10 ml Destain Solution for 10 minutes each with rocking.

• Equilibrate in 10 ml Electroelution Buffer and electroelute for 1 hour at 100 Volts in the same buffer.

• Recover approximately 500 ml and add 1 ml 6M guanidine-HCl in HEGK10.

• Dialyze against 500 ml 2 fold diluted guanidine-HCl solution for 30 minutes and repeat in 5 twofold steps.

• Dialyze against HEGK10 with PMSF and protease inhibitors. Concentrate and freeze in liquid nitrogen.