Anti-Tubulin antibody [B3] - Polyglutamylated (ab11324)

I have some technical questions. I would like to ask if the antibody is the functionally the same as the monoclonal antibody GT335 cited in the reference in your datasheet."Bobinnec Y et al. Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells. J Cell Biol 143:1575-89 (1998)." Actually, i would like to use the antibody to be the Centriole marker by immunofluoresence.

ANSWER:

Thank you for your enquiry regarding ab11324.

This antibody is clone [B3] and therefore it is not the same clone as [GT335] from the paper Bobinnec Y et al. You will however be able to use the antibody ab11324 in immunocytochemistry though we have not tested it in IF. The clone [B3] has been used in the following paper: Gagnon C et al. The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility. J Cell Sci 109 ( Pt 6):1545-53 (1996). PubMed: 8799841

I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

thank you for your mail. Do you know of other people using this antibody successfully (this batch)?. Is there a possibility for Abcam to test it, do you have a research division?. What are the chances that the 11324 was mixed up with another antibody? Again, I want to stress that we are truly interested in a commercial source for this antibody.

ANSWER:

Thank you for your email. We have sold other vials from this batch and have not received any other complaints (as I mentioned previously, this is the first complaint we have received regarding ab11324).

Ab11324 is originated outside Abcam (we do not make it in-house). We do have another batch in stock and I would be happy to send you a replacement vial from that batch if you're interested.

thank you for your mail. Here is the info

Our problem is that your antibody seems to recognize all of the microtubules in cells where there are only a few stable, glutamylated microtubules. Our control cells will have few stable microtubules. Taxol treated cells have many more stable, glutamylated microtubules. We use this to test you antibody. We compare the result of your antibody to that of a antibody against glutamylated tubulin (anti-GluTub) that we have from Dr. Cooper.

When we dilute your antibody down further, there is no change in the staining. I have to say it really does stain regular microtubules very nicely. Using secondary antibody only gave no staining.

We would very much like to have a commercial source for an antibody against glutamylated tubulin, that is why I hope you will be able to advise us about this.

We don't have the vial anymore, but received the order on january 25, 2005. There is no batch number on the packaging slip. The order number is 67521

Thanks again, and I look forward to hearing from you.

1. Please describe the problem (high background, no staining etc).

Problem: The antibody recognizes all microtubules, not just the glutamylated.

Our cells have hardly any glutamylated tubulin under control conditions. The 11324 stains all the microtubules very bright in this sample. In our taxol treated sample, there is a huge increase in the number of stable, glutamylated microtubules. The 11324 antibody may recognize that, but as is stains all the microtubules, we cannot see increase. We have one other anti-glutamilated tubulin antibody which we use as standard. Is shows little staining in our control (11324 shows a lot) and a lot in taxol treated.

2. On what material are you testing the antibody in IHC? We are testing immunofluorescence on C2C12 mouse muscle cell line. The cells are grown on glass coverslips. •Species? •Cell line? •Tissue?

3. How did you fix the samples? The samples are fixed for 6 minutes in ice cold methalol •Ethanol, methanol •Acetone •Paraformaldehyde •Other

4. Did you apply antigen retrieval step? no additional step •Enzymatic method •Heat mediated technique •Other

5. How did you block the unspecific binding sites? We block for 1 h at room temperature in 5% goat serum, 2 % bovine serum, 1% horse serum in PBS. The antibody solutions are made up in this buffer.

6. Primary antibody antibody 11324 at 1:1000, or 1:1500 or 1: 2000, incubated for 60 minutes at room temperature. The antibody has been stored in the refrigerator and freshly diluted.Wash is 3 times 10 minutes in PBS •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)?

7. Secondary antibody From Jackson laboratories, Goat anti mouse IgM, conjugated with TRITC, at 1: 200, incubated for 45 minutes at room temperature, followed by 3 washes in PBS for 10 minutes at room temperature. We have not tested this secondary with another primary. Secundary alone gives no staining. For detection, we use the fluorescence microscope

•What secondary antibody are you using? •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation, wash steps? •Do you know whether the problems you are experiencing come from the secondary? •What detection method are you using?

8. Background staining •Please provide an image of your staining

9. Which detection system did you use?

10. Did you apply positive and negative controls along with the samples? Please specify. Our negative control are cells that have not detectable glutamylated tubulin beyond the primary cilia at the MTOC. Our positive control is the taxol treated cells which have many glutamylated microtubules. This is detected with our other (non commercial antibody)

11. Optimization attempts •How many times have you tried the IHC? 2 times •Do you obtain the same results every time? yes •What steps have you altered? primary incubation time (shorter), diluting the antibody to 1:2000.

ANSWER:

Thank you for sending me details regarding your protocol and I'm sorry to hear that you are experiencing difficulty with ab11324. We have not received any other complaints regarding this antibody thus far. I don't see any problems with your protocol, and as you mentioned, the other polyglutamylated tubulin antibody that you are using is working fine. I can offer you a replacement batch of ab11324 to try. Please let me know if you would like to receive it.