Goat Anti-Rabbit IgG H&L (FITC) (ab6717)

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I am sorry for the delay in getting back to you. I now have further information to share in regards to how the Anti-Histone H2B antibody [EP957Y] (ab52599) has been used in staining of fixed cells.

The image presented on the datasheet of ab52599 shows the results obtained when using the antibody in the staining of Histone H2B in HeLa cells. This was achieved using acetone at -20°C for 5 minutes to fix and permeabilize the cells. The antibody has also been used successfully with fixation using 2% paraformaldehyde for 20 min and permeabilization with 0.1% Triton-X100 for 5 min. This is described in more detail in the following protocol:

http://www.epitomics.com/products/support/icc

I hope this information has been of help. I also wanted to bring to your attention a webinar we are hosting this afternoon on "Optimizing IHC/ICC results through careful experimental design" which may be of interest to you. More information on this can be found from the following link. It is free to attend.

https://www.abcam.com/index.html?pageconfig=resource&rid=15358

If you have any further questions please do not hesitate to ask.

Until then, I wish you all the best with your research.

Thanks for your reply. I am glad you found the information useful.

With the fixative you intend to use I do not think you will require permiabilisation. However, methanol is sometimes not suitable for some targets/antibodies. It may therefore need some optimisation in order to obtain the best results.

I am currently contacting the lab to see how this antibody was used in IF previously and what fixation was used. Once I have further information to share I will get back to you.

In the meantime, if you have any further questions, please do not hesitate to ask.

Thank you for contacting us this morning in regards to the anti-Histone H2B antibody (ab52599) and the Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC) (ab6717).

These antibodies should work fine in combination for the experiment you are currently planning. As discussed over the phone, we have quite a useful protocol guide which can be accessed from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13111

You can also request a hard copy of this from the link. This provides a good overview of the main antibody applications as well as providing troubleshooting should the results not be as expected. As you are working with a nuclear target, I would suggest introducing a permiabilisation step. For example incubating your samples with 0.2% triton for 10 minutes (unless you are using acetone or methanol for fixation). The antibody could be diluted in PBS with 0.1% tween as well as 0.1-1% BSA. We would normally recommend using overnight incubation at 4°C as this tends to result in a more specific signal.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Thank you for contacting us.

We have an isotype control of goat IgG, conjugated to FITC. The catalogue number is ab37374.

Click here (or use the following: https://www.abcam.com/FITC-Goat-IgG-Isotype-control-ab37374.html).

This is sold as "100 tests" (10ul per test). The concentration is 0.1mg/ml, and the volume is 1ml.

Please note that ab6717 has a different concentration (2mg/ml) and also a different concentration of the preservative sodium azide (0.01% as compared to 0.1% for ab37374, 10-fold higher). This may be an important consideration if you are staining live cells, as sodium azide can be cytotoxic.

Please let me know if you have any questions.

Thank you for contacting us.
According to the datasheet, the this protein is not expressed in most lymphoid cell lines: B-cells, T-cells and NK cells. Therefore, I would suggest to use a T-cell line like Jurkat lysate as a negative control.

https://www.abcam.com/Jurkat-whole-cell-lysate-ab7899.html https://www.abcam.com/Jurkat-whole-cell-lysate-ab7899.html.

As a "negative control" for WB I woul suggest to perform a non-primary control, which simply means skipping the promary antibody incubation.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

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I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Thank you for your reply and for returning the vial of ab6785.

The vial of ab6717 will be arriving tomorrow on the order ***.


Please let me know if you need anything else, and have a wonderful evening.