I am currently probing for CD63 tetraspanin, in exosome isolates, by western blotting. Thought I should include a loading control such as a housekeeping protein. Actin/tubulin/etc work for cells but which housekeeping protein works for exosomes? Any ideas?
i've tried beta-actin and they work fine, also try GAPDH. also remember when you probe for CD63, you have to keep the protein at its native form, otherwise the antibody won't recognize the protein. The same goes to other tetraspanins you might want to probe later, CD81 and CD9. None reducing western is the way to go. Good luck.
Hello Tommy. Thanks for your comment. Yes, I will be probing for other tetraspanins down the line. By non-reducing, you mean excluding the DTT/bME during sample prep? Will denaturing (SDS/heating) affect CD63 detection? Should I also worry about gel/buffer types? I use 4-12% precast Bis-Tris gels and MES running buffer.
Depending on the respective antibody, non-reducing conditions need to be used to analyze tetraspanins by WB. This indeed means using sample buffer without agents such as DTT/b-ME.
Depending on who you want to believe, heating might reduce signal intensities for CD63, but it's probably best to verify that yourself in your setup.
In our hands, CD63 is nicely detected using these procedures: lysis of EVs in non-reucing sample buffer (including SDS, though), incubation at RT for 30 min, separation of proteins on 4 - 12 % Bis-Tris-Gels using MES buffer, blotting onto nitrocellulose membranes.
Good luck!
Hello Dominik,
I have done some optimizations already and...
Yes:
1) bME and DTT are absolute no/no for CD63 and CD9 (the two tetraspanins I have worked with so far). The reducing agents completely wipe out CD63/9 bands.
2) denaturing with SDS is ok. No need for fully native preps.
3) 4 - 12 % Bis-Tris-Gels using MES buffer and dry transfer (x7min), on PVDF though (did not try nitrocellulose, but should be fine), worked well.
However, I did not see any significant differences between heating at 95oC x 5min, 75oC x 10min or just 37oC x 30 min. I guess because I optimized with a high expression positive control lysate (tip: platelets have tons of CD63 and some CD9; HeLa cells and human fibroblasts also express some CD63 but no CD9). Maybe 37oC x 30min or RT x 37min will be best when low yields are expected.
Thanks everyone. I hope this thread helps someone else with similar questions.
Happy Thanksgiving!!
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