Anti-CCR2 antibody (ab1668)

BATCH NUMBER 108763 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No staining

SAMPLE Human spleen and tonsil

PRIMARY ANTIBODY Tested dilutions of 1:100,1:200, and 1:400 incubated 1 hour at RT and O/N at 4C. Diluted in PBS and BSA.

ANTIBODY STORAGE CONDITIONS what is recommended

FIXATION OF SAMPLE 10% formalin

ANTIGEN RETRIEVAL 10mM citrate pH6.0 and 1mM EDTA pH8.0 in a decloaking chamber for 2 min. Let cool at RT for 20 min in hot buffer.

PERMEABILIZATION STEP Have Tween in my buffer (TBS)

BLOCKING CONDITIONS Biocare medical "sniper"---casein derrived non-specific background blocker. 10 min

SECONDARY ANTIBODY Jackson Labs---Donkey anti goat H&L HRP conjugated. Used at a dilution of 1:100 for 30 & 60 min. Made up in Biocare medical's van gogh yellow diluent.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Tried a new secondary at different dilutions 1:100 is about 5ug/mL

ANSWER:

I'm sorry to hear you are having a problem with ab1668.

We have had many orders for this antibody with very few complaints and I believe that modifying your protocol will help you get specific staining with this antibody. I would like to suggest the following modifications to your protocol in this order: 1) For the permeabilization step please try using 0.1% Triton X-100, 2)Instead of your blocking reagent, please try blocking in 10% normal goat serum with 1% BSA in TBS 2 hours at room temp, incubate the primary antibody at 1:200 in TBS with 1% BSA O/N at 4C, and incubate in secondary antibody for 2 hours at RT in TBS with 1% BSA, 3) Try no antigen retrieval next time, or try the heat mediated pressure cooker or enzymatic antigen retrieval instead, and 4) Applying the ABC signal enhancing step can improve the signal.

Please let me know if this helps and do not hesitate to contact us for further advice,

Customer received empty vial on order 80973 - FOCR created.

ANSWER:

I'm writing in regards to your order for ab1668. I apologize about the empty vial and I hope it was not too much of an inconvenience. I have created a free of charge replacement order for you that will ship today for delivery tomorrow. Please let me know if you have further questions.

I would like to know the specificity of this antibody and also ab13974. Does it recognize other ccr family members, i.e. CCR5 etc...? Or is it specific for just CCR2?

ANSWER:

Thank you for your enquiry about ab1668 and ab13974.

For ab1668 the information provided on our datasheet states that there is less than 50% homology between the peptide sequence used as an immunogen for this antibody with other chemokine receptors. For ab13974, I performed a BLAST search and found that there is less than 60% homology between this immunogen and other chemokine receptors and I will add this information to the datasheet. Based on this information I would predict that both antibodies should be specific for CCR2, but since the immunogen for ab1668 has less homology with other CCR's this one should be more specific.

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

What is the staining pattern of CCR2 ( ab 1668 ) in spleen tissue, nuclear or cytoplasmic? Any particular type of cells will stain up?

ANSWER:

Thank you for your enquiry.

The CCR2 antibody binds to the N-extracellular domain of human CCR2 on human spleen cells.

Customer would like to know what size band is expected with ab1668.

ANSWER:

I do apologize for the long delay in answering your question, I have only just heard back from the originator. The MW of CCR2 is 38 kD. If you have any further questions, please contact us again.