Anti-Cardiac Troponin I antibody (ab47003)

Thank you for contacting us.
I appreciate the time taken to submit protocol information. The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would also appreciate if you could please provide the order and batch details.
Reviewing this case, I would like to offer some suggestions to help optimise the results from these two antibodies in ICC/IF:
Cell fixation should be done under cold conditions. Using ice cold methanol, and optimizing it with other fixatives, such as 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.
For antibody ab24590 permeabilization is not required as the target protein is expressed in the membrane. For ab47003 I would recommend increasing the permeabilization conditions, by using more concentrated Tween 20 or 0.25% Triton X-100.
The unspecific signal obtained may be due to unspecific binding of the antibody as there has not been any blocking of unspecific binding sites. This could be improved by using 3%BSA for 30 min / 1 hour at room temperature.
Also, in order to facilitate the antibody to reach the target, incubation overnight at 4C normally gives better results than 1 hour incubation.
I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again and I will try to provide further help.

Thank you for getting back to me with the new results obtained.

It seems from the image shared that the staining is much improved with the changes in the protocol. The nuclear staining is much improved and the striations observed would be expected in the staining of this protein. In order to ascertain if there is any non-specific staining I would suggest the following:

1. Carry out a "no primary" control. This would involve staining the cells with the secondary antibody only at the same dilution as used before (1/40), then measure the fluorescence using the same parameters used for the images shared with me, i.e. using the rhodamine filters. This will allow you to see exactly how much of the non-specific staining is derived from the secondary antibody binding to the cells non-specifically. Ifnon-specific binding of the secondary antibody is observed Iwould suggest reducing the concentration of secondary antibody used to 1/200 and 1/500 to see if the results improve.

2. What buffer is being used to dilute theprimary antibody? I would suggest using 3% BSA in PBST.

3. Do you know what staining you would expect to get in human mesenchymal stem cells? Do you have any references where similar staining has been performed? Do you have access to a good positive control such as cardiomyocytes?

I hope these suggestions will be of help.


I look forward to receiving your reply.

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.