Hello,
I'm trying to costain cilia with acetylated-alpha-tubulin antibody (6-11b-1 mouse monoclonal) and something to mark the base of the cilium. My first attempt was with Anti-gamma-Tubulin (AK-15) which I thought would stain centrioles. Unfortunately my first attempt had lots of background staining with nothing that looked convincingly like a centriole. While thinking about ways to optimize my gama-tubulin staining, it occurs to me that somebody might already have a better way to do this.
If somebody could suggest a good antibody for this purpose, I would be very grateful.
Hi there. What about adenylyl cyclase 3 (ADCY3): santa cruz (sc-588). This one worked for my colleague in the lab I previously worked in, but I'm not 100% sure this is what you're looking for.
I'm looking for a basal body marker. AcIII will stain the whole cilium.
Ah my mistake, I thought it also extends through the basal body. Hope you find a good marker.
Jeff, once you know what proteins are fit as specific basal body markers, I can probably find you the best antibodies to such markers in the market. Yet another hurdle not to be underestimated.
Thanks Jan!
I'm well aware of that hurdle. I've actually got gama-tubulin working fairly well with some optimization of my fixation protocol. If you're good at finding the best available antibodies, that is a resource I'd definitely like to tap. There are a few other basal body proteins I'm interested in. I'll post them shortly. Also if you know of a better gamma-tubulin antibody than the one I'm using, that would be quite helpful. I need it to come from something other than mouse, as my acetylated-alpha-tubulin AB is from mouse.
Ideally I need something that works well in human and mouse and I image on confocal
TUBG1 is highly conserved between human and mouse, so no problem there. You seek antibodies fit for ICC, whilst majority of commercial antibodies only show WB. Some mabs are out there that might work, but they need a closer look into. A few rabbit antibodies..... Please contact me for further assistance.
Hi there,
I've been using anti-gamma-tubulin (GTU-88) as a centrosome and basal body marker for a period of time while performing IF staining.
The binding is specific with 1000 fold dilution.
Thanks Che-Wei Hu.
Unfortunately my acetylated alpha tubulin AB (is from mouse, so I couldn't use gtu-88 for costaining. I should have mentioned that in my question.
Jeff, you may consider conjugating a mouse mab to TUBG1 if you cannot find a good working non-mouse antibody. Of course the mab needs to be available as a purified antibody without carrier protein around for successful conjugation. Then MOM is no longer an issue.
Hi Jeff,
Have you found another basal marker antibody which hasn't produced in the mouse? I have the same problem, using mouse monoclonal acetylated tubulin and looking for a non-mouse basal marker antibody?
Try this one :
Sigma: Anti-γ-Tubulin antibody, T5192, Rabbit Polyclonal
Hi Elham,
I found several good basal body markers that worked for ICC but nothing gave satisfying results in IHC. In the 2 years since I asked this question, I've for the most part moved away from using acetylated tubulin to mark cilia because it is prone to artifacts. Now I mostly use Arl13b. I use NeuroMab clone N295B/66 if I need a mouse monoclonal. This is the cleanest staining cilia marker available. You can buy the same clone from abcam if you are in a hurry to get it. If you need rabbit poly, proteintech 17711-1-AP is excellent also. Unfortunately, arl13b isn't universally expressed in neuronal cilia. I don't have a good marker for that yet. Santa Cruz made an AC3 rabbit polyclonal (sc-588) that worked great for staining neuronal cilia. I've tried about a dozen other AC3 antibodies with no success. The logical conclusion is sc-588 stains something other than AC3 that sorts to neuronal cilia. Unfortunately santa cruz hasn't been allowed to make polyclonal antibodies for a few years now so I'm stumped. Acetylated tubulin has the obvious issue in neurons that AIS look a lot like cilia so tissue staining is a big mess.
Hope this helps. Good luck!
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