Anti-CCR11 antibody (ab1660)

Please see attached letter and images for further questions. Note especially that I would appreciate to see some of your images at a higher resolution, as the one on the internet is very hard to assess. In the quoted articles there are no image of tissue stainings that I am able to find. Please correct me if I am wrong.

Thanks again,

Q: In western blotting of wild type mice, a band is seen at 35kDa, is this CCR11? A: CCR11 has 351 amino acids with 3 glycosylation sites and 7 hydrophobic domains. 7TMs generally slower than other proteins of similar MW. The protein should run around 45 kD unless what they are seeing a large fragment following proteolytic degradation. Even in histochemistry all cells did not stain with the antibody. Since you are using a tissue lysate, we recommend to use 150ug of lysate to load on to the gels and Tris-Glycine blotting buffer containing 0.1% SDS and 20% methanol to transfer the proteins.

This is more or less what we have been doing. I have got a slightly different protocol now that I will try out.

Q: CCR11 knockout mice are still displaying a staining for CCR11. A: Are you getting staining with wild type mice in the same tissue? Is the staining pattern the same?

See attached images. Yes, exactly the same pattern.

We recommend to optimize staining with wild-type mouse tissue by titrating the antibody. 1:100, 250 and 500 dilution. Use the following buffer for paraffin sections: Dako buffer S-1700, pH 6, steam for 20 min, cool for 20 min). Then do the staining of paraffin sections from the knockout mouse at the same dilution and look for staining and differences in staining pattern as well. Use another antigen specific affinity purified goat antibody as control to make sure staining is not non-specific. Normal goat IgG may have specificity to epitopes conserved with mouse. This is what we have done, pretty muc. Dilutions 1:200 - 1:20000, a panel of different buffers including NaC pH 6.0.

You stated that mouse genomic DNA was sequenced to confirm mice were CCR11 knockout. Did you confirm by RT-PCR on lung tissues or by Northern Blotting to verify again that there were no correct size RNA transcripts that could hybridize with the probe?

I am not a molecular biologist by any means. The people that developed the mice are, though, and we discussed this quite a lot. They did say they used RT-PCR, so I am sure they will have done what you are asking.

ANSWER:

The lab was very impressed by the level of information you provided and would like to help as much as possible.

Their interpretation of the data sent is that the knock out mouse does not have deletion of the gene but a disruption of the coding sequence. That means some of the N-terminal sequence is in frame and thus producing protein containing N-terminal sequence but will have non-functional CCR11 receptor due to disruption of the coding sequence by insertions. The lab scientists recommended the following " please ask the Molecular Biologists who generated the knock-out mouse and did sequencing to confirm that coding sequence of CCR11 is completely deleted or disrupted. If disrupted, then the IHC images of N-terminal CCR11 protein seen make sense.".

I would be happy to provide you a refund if the gene has been completely deleted. I am also in the process of adding very nice staining images on the datasheet. I hope to have those soon there,

Thank you for your help, but our knock-out mice are knock-outs for CCRL1, not CCRL2. I have also talked to the scientist who gave us the tissues, and they have characterised them very well, and there should be no chance of CCRL1 in the tissues. The mice that were sent to us also underwent genomic sequencing, to make sure they were indeed knock-outs.

We also used heat-based techniques for AR, with a panel of buffers. I am preparing a more thorough overview of what we have done and questions I have, to send you.

In the meantime is there any chance of seeing the images that your lab has used as basis for their recommendations? I am not getting any staining above background in human tissues, but very distinct staining in mouse tissues. I can not get the antibody to work on cryosections. Have your lab managed that? Again, it would be very informative for me to see the images.

I would also like to know what size band you are getting with this ab on WB, and what material the WB showed on the datasheet of antibody ab32564 (rabbit monoclonal anti-CCR11) is done on (cells, tissue lysate or recombinant protein).

Thanks again, I really apprciate that you are helping us get to the bottom of this. Regards,

ANSWER:

Here is more information that I hope will be helpful.

Q: In western blotting of wild type mice, a band is seen at 35kDa, is this CCR11? A: CCR11 has 351 amino acids with 3 glycosylation sites and 7 hydrophobic domains. 7TMs generally slower than other proteins of similar MW. The protein should run around 45 kD unless what they are seeing a large fragment following proteolytic degradation. Even in histochemistry all cells did not stain with the antibody. Since you are using a tissue lysate, we recommend to use 150ug of lysate to load on to the gels and Tris-Glycine blotting buffer containing 0.1% SDS and 20% methanol to transfer the proteins.

Q: CCR11 knockout mice are still displaying a staining for CCR11. A: Are you getting staining with wild type mice in the same tissue? Is the staining pattern the same?

We recommend to optimize staining with wild-type mouse tissue by titrating the antibody. 1:100, 250 and 500 dilution. Use the following buffer for paraffin sections: Dako buffer S-1700, pH 6, steam for 20 min, cool for 20 min). Then do the staining of paraffin sections from the knockout mouse at the same dilution and look for staining and differences in staining pattern as well. Use another antigen specific affinity purified goat antibody as control to make sure staining is not non-specific. Normal goat IgG may have specificity to epitopes conserved with mouse.

You stated that mouse genomic DNA was sequenced to confirm mice were CCR11 knockout. Did you confirm by RT-PCR on lung tissues or by Northern Blotting to verify again that there were no correct size RNA transcripts that could hybridize with the probe?

Please do not hesitate to contact me if you have more questions or concerns.

CCR11 knockout mice are still displaying a staining for CCR11. In western blotting of wild type mice a band is seen at 35kDa, is this correct?

ANSWER:

Following extensive research we have discovered that there are two proteins called CCR11(the nomenclature has a confusing history) and that this antibody should be identified as identifying CCR11 and CCRL1 and not CCR11 and CCRL2, I have made this clearer on the datasheet.

Am I correct in understanding that the knockout mice are CCRL2 (CCR11), which would explain why ab1660 still give a staining with those mice?

I have found out that the antigen retrieval method used was heat mediated and added this to the datasheeet, I am still awaiting for information regarding the detected MW, my apologies.

I hope the above clarification on what type of CCR11 ab1660 detects has helped you understand your results, please do not hesitate to contact me if you have further questions,

What size band is detected with this antibody?

ANSWER:

Thank you for your enquiry. You should see a band at approximately 40 kDa. If you have any further questions, please contact us again.