Anti-PYK2 (phospho Y402) antibody (ab4800)

Thank you for contacting us and your interest in our products. I am sorry for the delay in getting back to you.

As we discussed over the phone, I have looked into how the Anti-PYK2 (phospho Y402) antibodies ab4800 and ab131543 have been characterized for their potential cross-reactivity with other phospho-proteins.

No specific experiments have been carried out to ascertain the cross-reactivity of either of these antibodies. However, the western results are based on total cell lysates which contains thousands of other proteins many of which are phosphorylated. As the Western blot images for these antibodies show, only one band was detected in the molecular range from 37 kDa up to 250 kDa, there is no other visible band.

In addition, with ab4800 the target band was competed out with the phosphorylated immunogen peptide indicating the band we see is the targe Pyk2 pY402. This supports at least in the cells (primary CDF cells, and SDF-1a treated Reh cells) we studied we did not see any cross reactivity with other proteins (phosphorylated or not phosphorylated).

From blast search of the immunogens used, the area does not seem to share homology with any other protein (that have been added to this databank) suggesting there should not be any cross-reactivity observed.

Thank you for contacting us. I do want to apologize for the tardiness of this response.

I have been in contact with the originator labs for ab42622 and ab60878 PYK2 proteins, in regards to whether these products are phosphorylated at Y402. Unfortunately neither group was able to confirm whether Y402 is phosphorylated at this site and each recommended that phophorylation state may be determined by performing a western blot with a phospho specific antibody such as ab4800 (https://www.abcam.com/PYK2-phospho-Y402-antibody-ab4800.html).

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 your enquiry. Ab4800 was tested in IHC cryostat sections in the following reference: Melendez J. et al (2002) J. Biol. Chem. 277(47):45203-45210. The authors prepared mouse heart sections fixed in 4% paraformaldehyde/PBS overnight at 4 degrees C. The next day, the hearts were subjected to progressive sucrose gradient from 10%, 20%, 30% at 4 degrees C for 1 hour at each step. Embedded in Tisse-Tek, frozen in melting isopentane floated above liquid nitrogen, and frozen blocks were stored at -80 degrees C Section 7 microns thick were mounted on Superfrost slides (Fisher Scientific Co), allowed to dry, and then stored at 4 degrees C until used within 1 week. For labeling, primary antibodies were applied to the sections and left overnight. The next day, the sections were washed, secondary antibodies applied to the sections for 1 hour, washed again, and then mounted in Vectashield (Vecyor Laboratories). Primary antibodies were detected by fluorescent secondary goat anti-rabbit IgG. We recommend using the antibody at ~1:50 dilution to 1:100 dilution. If you have any more questions, please contact us again.