Anti-Carbonic Anhydrase IX antibody (ab15086)

Hello, I am writing you re your Indirect ELISA kits and hope you can advise which kit buy and protocol to use. We have developed RCC mouse model using  RENCA mouse RCC cells over expressing the human CAIX protein. We would like to test/control for the possible presence of mouse anti human CAIX antibodies in the mouse sera. That is to say we would like to coat the plates with recombinant human CAIX protein, use OUR mice sera as primary Abs (as a “detector Antibodies”) and then follow with a secondary Anti Mouse Ab  HRP Conjugated/other detection conjugate. Could you possibly advise which kit buy and protocol to use. Thank you

ANSWER:

Thank you for contacting us.

Unfortunately we do not have any kit corresponding to the detection of the CAIX protein.

However, I would suggest to develop an ELISA assay using the CAIX protein ab114200 that would be coated on the plate. A control anti-CAIX, such as ab15086, for which the concentration is known, may be used to create a standard curve and to quantify the amount of anti-CAIX antibodies contained in the mouse sera.

ab15086 is a rabbit antibody. A mouse antibody would ideal, unfortunately, the only mouse anti-CAIX we have (ab107257) is not purified. It is an ascites surpernatant and the concentration has therefore not been determined.

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

Dear Colleques,

I would like to order antibody against CA IX (ab 15086). I need to use them for 150 formalin fixed, paraffin embeded sections.

Let me know about the amount I need to order, wheter they work with ENVISION FLEX+, HIGH pH, FOR AS, and about the price in total.

I need these information as soon as possible.

Best regards,

ANSWER:

Thank you for your patient. Unfortunately there is no number that I can provide to you as it is impossible to know for sure how many slides you will be able to stain with a vial. This will depend on the experimental conditions, the samples and how highly expressed the protein is, and what dilution you end up choosing. This antibody is recommended to be used at a 1:1000 dilution for IHC staining as this has worked well for our lab. I would recommend ordering a single vial and test it to determine what amount will be best for your assay rather than order too much or too little without first working with the antibody. The price for a single vial is €370.00 (without shipping costs). For more information about prices and shipping conditions, please contact our Customer Service department at: orders@abcam.com Regarding your second question, we haven’t tested this system in our lab, so I cannot assure whether it will work. However, it has a fairly normal range pH in the buffer (7-8) so most likely the functions best around that range and not at a high pH. I hope this information is helpful. Please do not hesitate to contact us if you need further advice or information.

Greetings-  

I would like to kindly ask you for any information on the following topic: my customer would like to purchase the ab15086 for IHC-P and he is asking what is the efficiency of this antibody for 1.5 mmx15mm fragment? In other words, how much this antibody he has to use to prepare 150 sections/fragments ?

Can he work on EnVision Flex+ visualization System with abcam antibodies?  

Thank you in advance for your help, I really appreciate it.   Best regards,    

ANSWER:

Thank you for contacting us.

The concentration which is recommended for performing IHC-P with Anti-Carbonic Anhydrase IX antibody (ab15086) is 1/1000-1/2000. Taking the more concentrated range, I have estimated that from the 100µl provided (and incubating with using 200 µl of diluted antibody per slide) a total of 500 slides could be stained. For 150 slides this would equate to a total of 30µl of the vial used. This is only an estimate and optimization with the individual tissue would need to be done.

In answer to your second question, although we have not tested our antibodies using the EnVision Flex+ visualization System, from having reviewed the product I seen no reason why it would not be compatible.

I hope this information has been of help. If you have any further questions please do not hesitate to contact us again.

I have only tried changing the preincubation step (antibody with blocking peptide) at different amounts of time (so, once overnight at 4 degrees and once for 30 mins at room temperature). The one overnight did help a bit but the bands that should be blocked were still quite bright.

To answer you questions:   1) What samples did you use (sample type, species)? I am using lysed keratinocytes infected with HPV 16

2) What antibody dilution have you tried? I have tried many antibody dilutions ranging from 1:200 to 1:10000 and I have found that 1:10000 gives me the best result (no background, but bands of interest still nice and bright).

3) What blocking reagent have you been using? The blocking reagent I use is 5% milk in 1XTBST

4) Which peptide dilutions have you tried? How long did you incubate the antibody with the peptide before the doing the Western blot?  For the peptide, I have only tried the suggested amount of 1ug/mL. As stated above, I have tried two different times; 30 minutes at room temperature or overnight at 4degrees.

Please let me know if it is obvious that I have missed something here or if I should just try a higher concentration of peptide or perhaps a longer preincubation at room temperature.

ANSWER:

Thank you for your reply.

It seems from your description that you are doing everything fine.

If I understand correctly, you are using the peptide at 1 ug/mL and the antibody at 0.1 ug/mL (1/10,000 dilution). At same volumes, the peptide is being used at 10-fold excess. Since this gave only a small effect in blocking, I would suggest trying the peptide at a 100-fold excess.

As for the incubation time: if overnight incubation at 4 degree worked a bit better, I would stick with this.

Should the increase in peptide not help, please let me know. If the peptide was purchased within the last 6 months, we can discuss options of replacement, credit or refund.

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

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Non-specific staining

SAMPLE human colorectal cancer

PRIMARY ANTIBODY ab15086 carbonic anhydrase IX

ANTIBODY STORAGE CONDITIONS just bought

FIXATION OF SAMPLE formaldehyde

ANTIGEN RETRIEVAL citrate

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

ADDITIONAL NOTES I can't answer most ofthe qustions above as I am not the individual who does the specific procedure. But the question is:

Using IHC, we are seeing very high staining of muscle. We see the expected membrane staining associated with CAIX staining in a few areas of the tumor, but surmise that the antibody is cross-reacting with something else in muscle. Using identical reagents (but different primary) we do not see any of this staininig. Are you aware of anyone else who has had this problem? I see no comment about it in the literature. All thoughts appreciated.

ANSWER:

Thank you for your enquiry. I enquired with our source for this antibody and there have not been any reports of this problem where the antibody is staining muscle. Ab15086 was characterized for use in IHC with renal carcinoma tissue and I would recommend that as a positive control.

If you can provide some additional details regarding the protocol that was used, we can further investigate this issue. An image would be helpful as well.