Anti-FGFR1 (phospho Y654) antibody (ab59194)

Thank you for contacting us.

I have conducted a search through our catalogue for antibodies against pFGFR1 and pFGFR4 which do no cross-react with the other FGFRs.

Ab59180 is specific against FGFR1 when it is phosphorilated at Tyr766. Similarly, ab59194 is specific against FGFR1 when it is phosphorilated at Tyr654, while ab52165 is specific against FGFR1 when it is phosphorilated at Tyr154.

These three antibodies do not bind to non-phosphorilated proteins. If you would send me the accession numbers or the exact protein sequences of other FGFRs that you are concerned with cross reactivity, I can align them with our immunogen sequence for sequence homology and possible cross reactivity.

We do have other alternatives (ab111124 and ab134043) against pFGFR1. However, these antibodies may also detect FGFR2, FGFR3 and FGFR4 phosphorilated at the equivalent sites. Although the antibodies have not been tested to determine if it detects these other endogenous forms, based on sequence homology reactivity would be expected.

Unfortunately, all the https://www.abcam.com/Search?Keywords=FGFR4+&fReset=1&pt=1&source=TopSearch&btnSearch=Search are against non phosphorilated forms, so I am afraid they would not fit your requirements.

Please do not hesitate to let me know whether you require a proforma, or any further assistance.

For diluting these antibodies for western blotting, we recommend PBS, 0.1% Triton X-100, with 1% BSA.

Thank you for contacting us.

I have received the following information from the lab:

1) This antibody reacts with target strongly when target is phosphorylated only on Y654 (phospho-Y654).
2) This antibody has weak reaction with target when both Y653 and Y654 sites are phosphorylated (phospho-Y653-Y654).
3) This antibody does not react with target when target is phosphorylated only on Y653.

We tested with three phospho-peptides: phospho-Y653-peptide; phospho-Y654-peptide; and phospho-Y653-Y654-peptide (double phospho sites).

The phospho-Y654-peptide blocked this antibody reaction, completely.
The phospho-Y653-Y654-peptide (double phosphos sites) blocked the reaction, partially.
The phospho-Y653-peptide did not block the reaction.



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

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab59194. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

Thank you for contacting us. Indeed, crossreactivity is always a very important information for an antibody. Sequence alignment and experimental data can help to predict/determine this. We receive these antibodies from a collaborating laboratory. I have asked the laboratory to know how the specificity has been determined and whether crossreactivity has experimentally been assessed. The laboratory had not assessed this crossreactivity experimentally. They have run a blast with the exact immunogen sequences (to which I have no access) and does on that basis exclude any possible crossreaction with other proteins than FGFR1. The regions from which the respective peptide immunogen of the antibodies are ab52165 (Tyr154), ab59180 (Tyr 766) and ab59194 (Tyr654). Please find here the sequence alignment for FGFR1 with FGFR2: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-091151-0127-47989414-oy For predicted crossreactivity with FGFR2: Comparison of the sequence with FGFR3 and FGFR1: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-103134-0638-15807362-pg Sequence alignment of FGFR1 with FGFR4: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-103350-0499-7232840-pg The sequence around Tyr154 has deletions in the FGFR2 and FGFR3 sequence when compared to the sequence of FGFR1, resulting therefore in a very poor alignment of that region. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us. The protocol is as follows: a. 293 cells were plated on flask or dishes containing growth medium (basic medium containing 10% serum). b. When the cells were at the log phase and 80-90% confluent, the culture media was drained. Wash cells with fresh basic media (no serum) once. c. Add fresh basic media to the vessels and starve the cells for 18-24 hours. d. Drain the cultured medium, then add fresh basic medium (about 4ml per T75 flask) to the vessels. e. Add insulin to the medium at 0.01U/ml. f. Cells were incubated at 37°C for 15 minutes in a CO2 incubator and then were used to prepare cell lysis. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry and your interest in our products. I have discussed your enquiry with my colleagues in the Lab and we think the antibody can detect both endogenous FGFR1 (phospho Y654) and the induced/stimulated FGFR1 at this modification site. We have only tested the antibody in 293 cells treated with insulin. I hope this will be usefulr for you.