http://www.sciencedirect.com/science/article/pii/S1534580711003571

see figure 5 in this paper. there is one band at 170 kDa for CD63. I think 200 kDa is possible. what you can do is that if your antibody was raised againsta  peptide immnuogen, then if you do competition study with one blot in presence and other blot in absence of this immunogen peptide then this band should selectively disppear. if the natibody was raised agianst whole protein then there is hardly anything you can do. In that case you can do a positive control of some cell lysates where it is alreadfy publsihed at what kDa the CD63 band appears. if your antibody can match those bands then you can take it that your 200 kDa band is for real.

Hi. I don't know CD63 protein. Are you sure CD63-glycosylated protein is in your samples?Do you have a positive control? Do tou have used it before? Did it work? If yes, I think the problem is the antibody and you can contact Santa Cruz to have a refund. The same thing happened to me with Abcam and they sent me another antibody batch.

Good luck, Bye.

No, I don't think my CD63 is glycosylated.  I think that it is appearing at the wrong size.  My marker is correct, and I have no other bands anywhere on the blots.  Technically perfect blots, with bands only in the lanes that I expect, except for the fact that they are running "too high".  

with santa cruz antibody, I have also found CD63 in plasma exosomes at 200 kda. but in urinary exosomes at 65 kda. polylactosamine extension of CD63 is known. Also in case of bigger vesicles such a smicroparticles, we have observed CD63 at more than 100 kda band ain addition to the band at usual place. What is the source of your exosomes...?

I extract my exos from Plasma and CSF.  I am not familiar with polylactosamine extension.  Will have to look into that.

http://www.sciencedirect.com/science/article/pii/S1534580711003571

see figure 5 in this paper. there is one band at 170 kDa for CD63. I think 200 kDa is possible. what you can do is that if your antibody was raised againsta  peptide immnuogen, then if you do competition study with one blot in presence and other blot in absence of this immunogen peptide then this band should selectively disppear. if the natibody was raised agianst whole protein then there is hardly anything you can do. In that case you can do a positive control of some cell lysates where it is alreadfy publsihed at what kDa the CD63 band appears. if your antibody can match those bands then you can take it that your 200 kDa band is for real.

We've had problems with the specificity Santa Cruz anti-CD63 antibodies that we have tested and only use Abcam mouse monoclonal MEM-259 (part number ab8219). Also, there is extensive glycosylation of CD63 and from our exosome preps we typically see bands between 40-65 kDa with the majority at 65 kDa. In cell lysates there is an even greater spread of MW species.

We also observed a smeared pattern for CD63 (40-65 kDa) both from cells and exosomes (Oskvold et al 2014) using the clone TS63 when we compared the profile in different B-cell lymphoma derived exosomes. It also appeared to be different smeared pattern depending on the origin.

HI Crista, I work a bit with the CD63 antibody and I have experienced better results using SDS-page gel,  you don't need that and they are more difficult to handle but  prepare the sample using sample buffer without DTT and cooking in the thermomixer at 37° for 10-15 minutes and you should see the classical smear given by this highly glycosilated molecule at the right MW. 

With Abcam mouse monoclonal (clone MEM-256), we typically see bands around 60 kDa from exosomes isolated from culture supernatants of EBV infected cells. 

I am getting band size of 70 kda after using CD63 sc-365604 antibody from Santa Cruz, the same problem !

The CD63 detection Ab we use is 10628D from Invitrogen which works very nice. 

I met the same problem as you，and if you have solved this problem?thank you

Hi Crista,

We have also noticed a wide variety of CD63 bands when performing WB on exosomes. This is highly dependent on your sample type, isolation method, and antibody. As others have stated, it is common to have multiple isoforms of the protein at different sizes based on their glycosylation state, though the 200 kDa band is odd. Your best bet is to ensure that your results are reproducible across different antibody vendors. If exosome quantitation is your goal, it might be useful to try a variety of different tetraspanins (e.g. TSG101, CD81, ALIX, CD9 to name a few). It may be that CD63 is not the appropriate marker for your starting material.

Best of luck,

Max