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Hello, I made the following changes: - Loaded 30 ug protein per lane (lane labeled HUVEC 1 was prepared using RIPA lysis buffer and lysate was sufficiently concentrated to load 30 ug; lane labeled HUVEC 2 was prepared from a dilute lysate, using a TCA precipitation) - Used phosphatase inhibitors (Phosphatase Inhibitor Cocktail A or Phosphatase Inhibitor Cocktail B from Santa Cruz) in addition to the protease inhibitor cocktail during cell lysis - Used a 12% SDS-PAGE gel, as compared to the NuPAGE Bis-Tris precast gels used previously I obtained the following results: Blot 1: monoclonal CTH, with phosphatase inhibitors Used 1:250 ab54573. Observed two strong bands that may correspond to the appropriate MW of the two cystathionase variants (see attached). Blot 2: monoclonal CTH, without phosphatase inhibitors Used 1:250 ab54573. Appeared similar to Blot 1. Blot 3: polyclonal CTH, with phosphatase inhibitors Used 1:250 ab80643. Observed several strong bands that do not seem to correspond to the desired 39 or 45 kDa MW. Blot 4: polyclonal CTH with HeLa positive control, without phosphatase inhibitors Used 1:250 ab80643. Detected several strong bands, but slightly different compared to Blot 3. Blot 5: monoclonal CTH antibody from Novus/Abnova Used 1:500 Appeared similar to Blot 1 and 2. Thank you for all of the suggestions. I am very satisfied with the performance of the monoclonal antibody (ab54573), but still have not been able to troubleshoot the polyclonal antibody (a80643) successfully. I appreciate your continued assistance! Sincerely,
Asked on Jan 09 2012
Thank you very much for keeping me up to date with the performance of the antibodies. I am very happy to hear you could obtain satisfactory results from ab54573. On the other hand it is regrettable ab80643 didn’t perform accordingly. I appreciate the time you have spent in the laboratory and understand your concerns. I can suggest you have regrettably received a bad vial of ab80643. As the order for this antibody was placed within the last 6 months, it is still covered by our Abpromise guarantee, and if you wish to, I can send you a free replacement for this antibody (from a different lot) a reimbursement or a Credit Note in compensation. I am very sorry for the inconveniences, and I will be happy to assist you with this problem, and give a quick and efficient solution. Therefore I look forward to hearing from you letting me know how you would like to proceed. Have a nice day.
Answered on Jan 09 2012