Question (31030) | Anti-beta III Tubulin antibody [TU-20] (ab7751)

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Question

 Dear Madam/Sir,   Thanks for sending me the new vial of the neuron-specific tubulin beta III antibody, it arrived on Monday. In the meanwhile I repeated my experiments and can confirm you that your antibody works a lot better than the previous vial I bought from you. Thanks!   Concerning the experiment I’m currently performing, I want to ask you some expertise if possible. I attached a file to this mail containing a blot of the immunoprecipitation experiment I performed yesterday with the new vial of antibody. This IP was done on 1250 µg mouse brain tissue with a range of antibody concentrations (1, 3, and 5 µg antibody). As you can see the IP works properly for the reaction with 5 µg antibody, but there is still a lot of tubulin in the supernatans. Do you know if there is something I can do to increase the amount of tubulin that can bind to the antibody+beads , and as such I can pull down more tubulin out of my reaction?   Thanks in advance .    

Answer

Thank you for your feedback. I am happy to hear that the replacement vial provides good results. In order to increase the amount of tubulin that is IPed, I have a couple protocol tips that might be worth trying: 1.) Tubulin is a very abundant protein and you are using a high amount of tissue and 5ug of antibody might be saturated without catching all the tubulin. I therefore suggest to increase the amount of antibody. 2.) It is possible that the binding of bead-antibody and tubulin is not optimal. I suggest to increase the incubation times and to make sure that the samples are under gentle agitation or rotation. Since there are two possibilities to incubate: (A) beads and antibody first or (B) antibody and sample first, I suggest to try both to see if the binding can be optimized. 3.) I can also recommend to check if the buffers (if using naive buffer, make sure the protein is not denatured during the IP) and proteinase inhibitors are in working order to prevent denaturing or degenerating proteins and antibody. I hope this information is helpful. Please do not hesitate to contact me with any further questions in this matter.

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