Santa cruz antibodies work at 1:500 dilution. Try dilution in 5% milk in PBST/TBST. Incubate overnight in shaker at 4 degree C.

Hi,

For CD63 detection by WB the clone TS63 works well. In terms of CD81 detection the clone M38 is a nice alternative what has worked well for me. Finally, the CD9 clone we have used extensively is the clone TS9.

kind regards

ketil

Hi, for detection of targets on western blotting its important to optimize and standardize the cell growth conditions to allow cells to secrete similar amounts of exosomes each time (possibly also exosomes of the same type - could be that e.g. stress induces release of other subpopulations of exosomes?). then its critical to optimize and standardize exosome harvest conditions so that one collect similar amounts of exosomes for each harvest.. Normalization of the input for WB is a topic for discussion and as far as I know there is no concensus yet on this even though protein measurments have been suggested (we have performed such protein measurements on all our preparations but it does not seem to correlate well with signals we obtain in flow using Dynabeads for exosome pull down). Light scattering analysis using instruments such as the NanoSight has also been suggested. As the amount of input often is low is recommended to maximize the amount loaded on the gel. In our hands wet transfer to PVDF seems to work best followed by using the right Ab clones  such as the ones we have tested extensively on such material in combination with a sensitive detection system

Feel free to contact me for more details at [email protected]

ketil

I'm using the CD63 from Santa Cruz.  It only works for me when I run a native gel and non-reducing conditions.  The only problem is, that it appears too big! Other than that, you know.....

I have no experience with CD81, sorry.

Good luck!

Thanks for your answer.

Could you tell me which SCB antibody clone have you been using?

Around which molecular weight do you get CD63 ?

I use CD63 H-193 right now.  The data sheet says 43 kd, but I only get bands at around 200kd.  I'm still working on it.   I have found that loading less protein than normal is better, but the band is still too big.  I load about 10 µg/lane.