Question (64099) | Anti-Aurora A antibody - Centrosome Marker (ab12875)

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Question

We recently bought this antibody and I experimented a major problem when using it for western blotting. I detected two bands, one between the 100 and 150 kDa molecular weight markers and one at a size below a 31 kDa marker. Unfortunately, this antibody is supposed to detect Aurora A and according to the product page of this antibody, it should detect a band migrating around 48kDa. I attached an annotated image of the result I obtained. To test this antibody, I produced a whole cell lysate of HCT116 cells (human colon carcinoma cells). 10ug (lane 1) and 20ug (lane 2) of proteins were loaded on a 8% SDS-PAGE gel. Gel was run for 1 hour and transferred at 100V for an hour and a half onto a nitrocellulose membrane. The membrane was blocked for one hour at room temperature with 5% non-fat milk in PBS-tween (0,05%) and was then incubated with the ab12875 antibody for 1 hour at room temperature (1/500, diluted in 1% non-fat milk in PBS/tween). The membrane was washed 3x 5 minutes in PBS-tween and was incubated for 45 minutes at room temperature with an anti-rabbit coupled to HRP (Dako, cat num: P0448) secondary antibody diluted at 1/5000 in 5% non-fat milk in PBS-tween. The membrane was washed 3 times for 5 minutes in PBS-tween. To reveal the bands, the membrane was incubated for 1 minutes in western lightning plus reagent (perkin elmer, cat no: NEL105001EA). The bands were detected by measuring chemiluminescence using a chemi-doc MP apparatus (Biorad).

Answer

Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Can you tell me a bit more about how you prepared your cell lysate? What kind of lysis buffer did you use? We would recommend NP-40 or RIPA buffer.

Are you including protease inhibitors in with the sample preparation? I see that the samples are denatured, but are they reduced as well with b-mercaptoethanol or DTT? Did you boil the samples for 10 mins?

Do you have access to a higher percentage gel? You may want to try a 12% gel for a protein below 50 kDa.

For transfer of small proteins (< 100 kDa), consider removing SDS from the transfer buffer and keep the methanol concentration at 20%.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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