Anti-MMP13 antibody [VIIIA2 ] (ab3208)

I purchased antibody MMP-13, cat# ab75606, and stained cell NUCLEI well in cartilage for rat bone tissue just as your picture posted on the web.  But also I looked at cat # ab3208, the antibody stained breast tissue as CYTOPLASMA satin. My question is; Does different clone of MMP-13 satins different part of cells? If I would like to see cytoplasma satin, Should I order ab3208?   Thank you very much for your help.

ANSWER:

Thank you for contacting us.

This protein is secreted into the extracellular space where it is cleaved and activated by proteases. Unless the area of the protein that an antibody recognizes is in a more accessible conformation in the cytoplasm, I see no reason why different clones should not recognize the protein where ever it is expressed. Have you attempted using an antigen retrieval step in your staining? This often helps to unmask epitopes and allow better recognition by the antibody.

Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

I have a question about anti MMP13 antibody [VIIIA2] (ab3208). Can ab3208 detect osteoclasts?

ANSWER:

Thank you for your enquiry. There is a reference that an antibody with same clone as ab3208 got positive result against osteoclast (PMID: 15442036).

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

BATCH NUMBER 344746 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No staining

SAMPLE paraffin embedded rat tibial tissue and human palcenta as control, all are cut in 5 microns

PRIMARY ANTIBODY MMP 13, mouse dilution 1:50 Room temperature, incubated for 1 hr and also tried overnight incubation in 1:500 dilution. Wash 3 times in

DETECTION METHOD aside from the kit mentioned above. I used Jackson Immunology Peroxidase Conjugated Strepavidin, that has crosreactivityt ot alomost all species inlcuding mouse,rat,human, donkey and hamster.

POSITIVE AND NEGATIVE CONTROLS USED No known positive control used except for the sample used placenta and no primary antibodies applied to a negative control

ANTIBODY STORAGE CONDITIONS aliquoted in 10 ul. kept at 4 degrees celcius refrigerator

FIXATION OF SAMPLE rat tibial fix in 4% paraformoldehyde, human placenta in 10% NBF

ANTIGEN RETRIEVAL Dako Antigen Retrieval Sloution (S-1700) using Dako pressure cooker and steaming for 20 minutes and cooling for 20 minutes.

PERMEABILIZATION STEP 3 % Hydrogen peroxide in distilled water and aslo did a 3% hydrogen peroxide in methanol.

BLOCKING CONDITIONS Use a Power Vision Poly HRP Detection Kit for Immunovision. The kit components 1. Pre-antibody Blocking (Primary dilueent,ready to use) 2.Post antibody Blocking (Polymer penetration enchancer) Ready to use 3.Poly HRP anti Mouse/Rabbit IgG (Ready to use) 4.1 drop of DAB solution A and B added to .93 ml of deionized water. other method tried: using 1% BSA as blocking solution and also using universal blocking solution from biogenex ( Casein in PBS)

SECONDARY ANTIBODY aside from the kit mentioned above, I used Jackson Immunology Donkey anti mouse at 1:500 dilution for 45 minutes.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have not altered the steps at all.

ADDITIONAL NOTES I've noticed that my tissue has lots of background staining when I used antigen retreival technique on Powervision kit. and without antigen retrieval it has a cleaner background but no staining at all. Please provide me images and your protocol that have been done on this antibody and the control used. Is there a possibilty to purchase a positive control from you.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab3208 in immunohistochemistry. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

I have looked at the protocols you used and have a few questions and suggestions that might help you determine the cause of the no-staining results. I would therefore appreciate if you can please clarify the following items.

- can you confirm that the second antibody and blocking solution you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working.

- is the diluent from Dako performing well in other experiments? What is the dilution for the secondary antibody?

- we would normally suggest doing antigen retrieval with Tris/EDTA pH 9.0 buffer as it is suitable for most antigens. Sodium citrate pH 6.0 is also widely used. Can you confirm that the antigen retrieval solution you are using also works well in other experiments?

- Can you confirm that after addition of the secondary antibody, the ABC complex was made and left to stand for a minimum of 30 minutes? This is the length of time that the complex takes to form.

- Please use PBS instead of distilled water in your endogenous peroxidase inactivation solution. Methanol is commonly used when handling frozen sections. All of our products used a general protocol which can be found on the website. If any special handling is required, it will be mentioned on the individual product datasheet. As stated on the datasheet, the positive control for this product are SW1353 cells, placenta, bladder, breast, and ovarian carcinomas.

I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order details (purchase order number, shipping address/purchasing agent, etc.). Also please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

Please, can you tell me a positive control for IHC paraffin staining? Thank you very much!

ANSWER:

Thank you for your enquiry. The following tissue is recommended as a positive control for use with ab3208: Placenta, and also bladder, breast, and ovarian carcinomas.

If you have any additional questions, please contact us again.

This antibody recognizes the pro and acive forms of MMP 13 - what size band(s) does it detect in Western blotting? Also, what dilution is recommended for WB?

ANSWER:

Thank you for your enquiry.

I contacted the originator of ab3208 and was informed that this antibody is not suitable for application in Western blotting. It does work in Immunohistochemistry (Formalin-fixed paraffin-embedded sections) and IP. Our datasheet did say that ab3208 was tested for application in IHC and IP and not yet tested in other applications. I have added to the datasheet that ab3208 is not suitable for WB.

I assume that when you purchased the antibody that you were under the impression that it had not yet been tested in Western blotting (which was stated on the datasheet). Under these circumstances, I can offer you a credit note or I can send you an alternative antibody of your choice, if you are interested.