Chemiluminescence Detection with Amersham ECL, Amersham ECL Prime, and Amersham ECL Select

1. Dilute the primary antibody in PBS-Tween or TBS-Tween.
   
   
2. Place the membrane (protein side up) in the primary antibody solution and incubate with agitation for 1 h at room temperature or overnight at 4 °C. Always refer to manufacturers’ recommendations.
   
   
3. Wash the membrane three to six times in PBS-Tween or TBS-Tween for 5 min per wash or according to manufacturers’ recommendations.
   
   
4. Place the membrane in the secondary antibody diluted in PBS-Tween or TBS-Tween and incubate with agitation for 1 h at room temperature or overnight at 4 °C.
   
   
5. Place the membrane in washing solution and wash four to six times for 5 min per wash.
   
   
6. Continue with detection as recommended for the selected detection reagent and imaging system.

Amersham ECL, Amersham ECL Prime, and Amersham ECL Select detection systems require very little antibody to achieve a sufficient sensitivity; therefore, the amount of antibody (primary and secondary) used in the protocols can be minimized. Smaller quantities of antibody-buffer mixtures can be used by scaling down the protocol and performing the incubations in sealable plastic bags.

Figure 4.4 shows typical Western blot results using Anti-GST Antibody.

Figure 4.4. Western blot of E. coli lysates containing GST-tagged proteins. For detection, Anti-GST Antibody, anti-goat IgG alkaline phosphatase conjugate, and DNB/nitro-blue tetrazolium chloride (NBT) enzyme substrate were used.