This antibody comes with an Incubation Solution which should be used as the diluent for Western blot (the antibody should not be diluted in milk for incubations). The lab has found that diluting the antibody in this solution gives optimal results. Dilute the antibody to 1 ug/mL in neat Incubation Solution before use.

The following is a quick reference procedure guide for Western Blot, specific for ab110322:

1. Solubilize samples in SDS-PAGE sample buffer.

2. Heat sample to 37°C for 30 minutes.

3. Separate proteins on Tris-glycine 10-22% gradient gel 150 V ~ 2 hrs.

4. Soak gel in 50 mL CAPS buffer for 30 minutes.

5. Transfer to 0.45 µm PVDF membrane in CAPS buffer for 2 hrs at 0.15 A CAPS buffer : 10 mM 3-[cyclohexylamino]-1-propane sulfonic acid, pH 11 w/NaOH, 10% methanol ice cold throughout.

6. Block membrane for 3 hrs - overnight in 5% milk/PBS 4°C.

7. Wash blot 3 times for 5 mins PBS/0.05% Tween-20 with agitation. Followed by a final rinse in PBS.

8. Antibody diluted to concentration specified in supplied incubation solution for 2 hrs.

9. Wash blot 3 times in PBS/0.05% Tween-20 for 5 mins.

10. Incubate blot with secondary anti-mouse antibody diluted according to manufacturer’s instructions in 1% milk/PBS for 2 hrs.

11. Wash blot 3 times in PBS/0.05% Tween-20 for 5 mins. Rinse blot in PBS.

12. Develop Blot: For alkaline phosphatase we recommend BCIP/NBT. For horseradish peroxidase we recommend ECL+.

Previously labelled as Adenine Nucleotide Translocase 1. 

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