Anti-Isocitrate dehydrogenase antibody (ab85532)

Dear Sir.,
We bought your antibody against IDH1 (rabbit polyclonal, Cat no. ab85532) and we got some questions about the antibody dilution. So, we receive a vial with 50ug (in +/-50ul) at concentration of 1mg/ml.
For IHC-P it is recommended a concentration of 10ug/ml...
Therefore, we understand we need to dilute x5 my ab, is it right?
If so, I need to add 200ul of pure water or PBS and this will give me 250ul of IDH1 at 10ug/ml.
The issue is that if is correct, I just have antibody for 1 -2 slides, since I have to pour 100ul of antibody per slide...
I think we are wrong!!
Sorry for the inconvenience.
Other issue is that we looked at your website and we found another ab mouse monoclonal (Cat no. ab117976 - clone 2H9 at 100ul/ml)... This monoclonal antibody is better than ab85532)?
Thank you for your kind attention,

ANSWER:

Thank you for contacting us.

As I understand it, if the recommended IHC working concentration for this antibody is 10ug/ml, and 100ul are needed for slide, then you would need 1ug of antibody per 100ul of diluent, that is, 1ug of antibody per slide.

As 50ug of antibody you would then have enough antibody to stain 50 slides, if I am not mistaken.

Regarding antibody ab117976, it is not a better antibody, it’s a different one against the same target. The main differences between both are the immunogen used to produce it and the clonality. Ab117976 is a Mouse monoclonal, whereas ab85532 is a Rabbit polyclonal.

If you want to have a look at the advantages and disadvantages of using polyclonal and monoclonal, you can have a look at our protocol’s site:

http://www.abcam.com/index.html?pageconfig=resource&rid=11269&pid=11287

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Customer requested the information regarding manual quantification of antibody signal in IHC-P.

ANSWER:

Thank you for contacting us.

I would say that this sort of experiment is very-very tricky. We unfortunately do not have any protocol available at the moment for manually IHC staining quantification with antibodies. I would suggest ploughing through the publications and finding if there are scientists who are using similar kind of method.

http://www.histochem.org/archives/photoshop.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1810239/

http://www.ncbi.nlm.nih.gov/pubmed/21370029

Have a look at these articles and check which one could help you to set up the experiment.

I hope this will help.

Does this product detect the wild type or mutant protein?

ANSWER:

Thank you for your enquiry.

This antibody was raised against isocytrate dehydrogenase purified from porcine heart. According to our collaborator who developed the antibody, they have only tested it against wild type isocytrate dehydrogenase. It has not been tested against any mutant forms of the protein. Therefore, we do not know if it could also detect the mutant form. Please note that this antibody is thought to recognize both the cytosolic and the mitochondrial form of the protein.

Also, the tissue shown in the image is from IHC using FFPE human heart.