Question (52862) | Anti-Cardiac Troponin I antibody (ab47003)

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Question

Dear technical team,

Our customer knows this protein is in cytoplasm ofcells buther datashowed in nucleus.
So she would like to know if this protein can be detected in nucleus.
Please check this.

Best regards

Answer

Thank you for taking time to complete our questionnaire and sorry for the delay in getting back to you. 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.

We would not expect the kind of nuclear staining observed in your experiments. Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab47003 and obtain the staining expected.

1. I would suggest altering the blocking used to 3% BSA instead of the serum. If you want to use serum then it should be from the species in which the secondary antibody was raised, in this case Donkey. BSA has been successfully used by several of our customers, one of which is illustrated from the following link:

https://www.abcam.com/index.html?datasheet=47003&tab=abreviews&intabreviewid=30342

In the blocking buffer I would also introduce 0.3 M glycine. This will bind any free aldehyde groups that could otherwise bind the primary or secondary antibody potentially leading toa high background.

2. I would also suggest reducing the concentration of the primary antibody used, from 1/100 to 1/400.

3. I would suggest checking the non-specific binding of the secondary antibody. This can be performed bu incubating the slide with the secondary antibody following the normal blocking step and viewing the slide as normal using the Rhodamine filter. If this is contributing to the non-specificity I would suggest reducing the concentration from 1/50.

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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