Anti-CD63 antibody [MEM-259] (ab8219)

Dear Technical,
I have used this antibody for Westerns and EM with good results. It is of
interest to know whether the epitope is known or, particularly, whether the
epitope is intra-or extracellular.
Kind regards

ANSWER:

Thank you for your enquiry.

I can confirm that the epitope for ab8219 CD63 antibody [MEM-259] has been mapped to the extracellular region of the CD63 protein.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

so for ab8219 you are not aware of any robust and reproducible Western data, but with ab59479 based on your experience its guaranteed that Western will work fine. You think depending on the cell line (and we'll also use exosomes from blood) we'll see several bands in the range 30-60 KDa?  We purchased both antibodies from abcam already, but Western conditions are not optimized yet. With 8219 we see one band ~ 42 KDa, another > 180 kDa. For ab59479 we see band ~40 KDa, just one faint band. Both gels were denaturing, non-reducing conditions. thanks,

ANSWER:

Thnak you for contacting Abcam.

In regards to ab8219, I have had the chance to talk with the lab. According to them this antigen is indeed very variable on WB of various material. I am sending the pattern they obtained on non-reducing K562 cells (see image CCE3541736_MEM-259WB.jpg), however observing more heavier bands using other material would be also a reasonable result.

I have attached information given to me by the lab producing ab59479 (see remainng attached). These include a puplication which has used the product in Western blot as well as a Powerpoint file which contains lab testing results.

I hope this information is helpful. Please do not hestitate to contact me if you have any questions.

Dear Sir/Madam: We need an anti-human CD63 antibody, to be used for Westerns. Specifically, we plan to detect CD63 protein from the human exosomes isolated from blood and cell culture media. Which antibody do you recommend- ab8219, ab59479 or other? PI sheet for Ab8219 anti-CD63 says that specificity is CD63, 25-26 kDa (or larger if glycosilated); But PI sheet for Ab59479 that is also anti-CD63 says that specificity is LIMP antigen, a 30-60 kDa (smear) protein; Thank you very much!

ANSWER:

Thank you for contacting us.

CD63 antigen is also known as LIMP and LAMP-3. While unmodified protein is about 25kDA, this is heavily glycolylated. AB59476migrates like a smear due to a very complexe N-glycosylation. I would recommend this product because this real world information is available whereas the data for ab8219 appears to be based on theory and not practise.

Please remember that differences exist between the cell lines (e.g. K562 givesbetter migration than HEL).

It is important to remember that this antibody (as well that all theanti-tetraspanine antibodies) must be used under non-reduced conditions.



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

Thanks again for looking into my inquiry from the other day. What I’m most interested in are the incubation times, blocking, washing times, amount of starting material, and amount of final primary antibody you guys use for quality assessment.

ANSWER:

The antibody was validated for western blotting using these conditions:

Sample: K562 cells (24 millions, lysed with 1% laurylmaltoside in 700 ul), non-reducing SDS, denatured by heat for 5 min.
Blocking: 5% milk, 4oC overnight
Primary antibody: diluted 1:100 in blocking solution, room temperature, 60 min.

I hope this is helpful. If you have any questions, please do not hesitate to contact us.

I am interested in protocol tips for using ab8219 in western blotting

ANSWER:

I am still in the process of looking into protocol information for using ab8219 in western blotting. I just wanted to let you know there is a reference using ab8219, noted under the 'Specific references' tab, that did use this antibody in western blotting. I thought this may be useful for you. Here is a link to the article:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817492/?tool=pubmed

I'll follow up with you as soon as I have some additional information.