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

Extracted from GST Gene Fusion System, GE Healthcare, 2014

  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.

Refer to GE Healthcare Western Blotting Handbook (28-9998-97) for further information on optimization of antibody concentration for Western blotting.

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.

Western blot of E. coli lysates containing GST-tagged proteins.

Fig 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.

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