Anti-CXCR4 antibody (ab7199)

Inquiry: I have a couple of questions regarding the CXCR4 antibodies for use on mice tissue. You have 2 products: ab2074 and ab7199. What is the difference between the two and which seems to be more effective (i.e. for immunoblotting and IHC on paraffin embedded tissues)? Also, when I gather tissues from mice, I would like to include an internal positive control. Which tissue would you recommend that can be used as a positive control for CXCR4 immunohistochemistry? Thanks.

ANSWER:

Thank you for your inquiry.

We would guarantee both ab2074 and ab7199 to work in mouse samples in IHC-P and WB. Both are purified and are 1mg/ml. The ab2074 is raised against a human peptide sequence and the ab199 is raised against the full length rat recombinant protein. Please note that ab2074 has a recommended specific protocol:

http://www.abcam.com/assets/popups/popup_datasheet_protocols_printer_friendly.cfm?pid=901&intAbId=2074

Oftentimes customers will choose an antibody that has the most references and Abreviews. Out of the two, ab2074 ismore heavily referenced and we have more IHC-P and WB data.

http://www.abcam.com/CXCR4-antibody-ab2074-references.html

http://www.abcam.com/CXCR4-antibody-ab2074-reviews.html

As for a positive control, we would recommend mouse bone marrow or skeletal muscle. Swiss Prot lists tissue specificity as: Lymphocytes, macrophages, neutrophils, microglial cells and astrocytes. Found in spleen, thymus, bone marrow, lymph nodes and, at lower levels in brain, small intestine, stomach and kidney. CXCR4-A is predominant in all tissues tested. During embryonic development, high levels are detected in the endothelium of developing blood vessels and in many regions of the developing brain including the olfactory epithelium, olfactory bulb, hippocampus, cerebellum and spinal cord.

http://www.uniprot.org/uniprot/P70658

In case you are interested, we sell mouse skeletal muscle slides as ab4609.

http://www.abcam.com/Skeletal-muscle-mouse-normal-tissue-slides-ab4609.html

I hope this information helps. Please contact us with any other questions.

I am the one who made the complaining about the CXCR4 antibody. I did try all different ways as you mentioned below and the staining didn’t show positive. Because I have tried two different antibodies from you company and did not get result. I wasted too long time and too many tissues on this staining. Would you please refund me for this antibody?  I have to purchase from other company.  

ANSWER:

Thank you for your reply. I apologize for the error with your name, as it seems that we had the incorrect name in our system for some reason. I have updated your account with the correct information.

Your credit note ID is ***. The full purchase price of $394.00 (including shipping) will be refunded to your credit card pending approval from our accounting department.

I am sorry that these CXCR4 antibodies did not work as expected, but if there is anything else that we can do for you please let us know.

No staining on rat heart tissue sections.

ANSWER:

Thank you for your phone call earlier this week and for letting us know about the trouble with ab7199. I am sorry that this replacement antibody is also not working as expected. IHC was added to our datasheet as a tested application based on positive customer Abreviews and publications using this antibody, so we don't have an in-house IHC protocol for ab7199. However, I do have a couple of recommendations that may help improve the staining results. If you are incubating the sections with the primary antibody for one hour at room temperature, you can increase the incubation to overnight at 4C. In one of the positive Abreviews for IHC, the user reported using an enzymatic antigen retrieval, so that may help improve the staining as well. Additionally, you can decrease the blocking time and/or dilute the CAS block in TBST. If the antibody is diluted in CAS block, try diluting it in TBST with no blocking solution. The replacement antibody is covered by our Abpromise guarantee, so if the results do not improve we would be happy to issue a credit or refund. Please let me know if you have any questions or if there is anything else that we can do for you.  

Does this antibody require antigen retrieval of formalin-fixed, paraffin-embedded sections? I noticed that one abreviewer used protease treatment, but I'm wondering if this is required, or if we can use heating in citrate or no AR at all.

Also, what staining pattern should we get on mouse spleen?

ANSWER:

Thank you for your enquiry.

I do not have any specific recommendations for this antibody, as the information that this antibody works in formalin-fixed paraffin-embedded sections comes from a customer review. I am not sure why enzymatic antigen retrieval was chosen, whether heat-mediated was tried first and did not work, or if this was the first method tried. If you go to the Abreviews tab on the datasheet (a link is included below), you can click on the reviewer's name to send an email to him and enquire about the reasoning behind using enzymatic vs. heat-mediated.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

We are looking for CXCR4 Ab for FACS in rat. Your ab7199 has been used in this application in your ref: "fischer AM et al. Regulation of CXC... tyrosine kinase ITK. J Biol Chem 2004; 279: 29816-29820" but the use of ab7199 for FACS is unclear in your datasheet. Could you please give me some more details ?

ANSWER:

Thank you for pointing out this discrepancy. I have added FACS to the datasheet as a tested application.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.